USE OF SECOISOLARICIRESINOL DIGLUCOSIDES (SDGs) AND RELATED COMPOUNDS FOR PROTECTION AGAINST RADIATION AND CHEMICAL DAMAGE

ABSTRACT

The invention provides compositions and methods for radioprotection and chemoprevention using therapeutic and prophylactics methods of using (S,S)-SDG (R,R)-SDG, (S,R)-SDG (R,S)-SDG, SDG, SECO, EL, ED, analogs thereof, stereoisomers thereof and other related molecules.

GOVERNMENT INTEREST

This invention was made with government support under Grant Numbers R01(CA133470), 1P30 ES013508-02, RC1AI081251, and 5-P30-CA-016520-34S2awarded by the National Institute of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

Provided herein are compositions and methods for radioprotection andradiation mitigation and for chemoprevention, such as fromcarcinogen-induced lung cancer and mesothelioma or from hypochloriteions, using secoisolariciresinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), stereoisomers thereof,metabolites thereof, and analogs thereof.

BACKGROUND OF THE INVENTION

Ionizing radiation produces a wide range of deleterious effects inliving organisms. Humans are exposed to radiation as an occupationalhazard, during diagnostic and therapeutic radiographic procedures, whenusing electronic devices, from background radiation of nuclearaccidents, during air and space travel, as well as from prolongedexposure to the sun (e.g., sun bathers or outdoor workers). Exposure tonatural radiation can occur in many forms: natural resources such asair, water, and soil may become contaminated when they come in contactwith naturally-occurring, radiation-emitting substances (radionuclides);radon is one such common source of natural radiation. Current globaldevelopments have additionally established terrorism as a dangerousmeans by which potentially large numbers of people can be exposed tolethal amounts of radiation. It is, therefore, of high importance toidentify agents that can be administered before and during exposure toradiation (i.e., radioprotective agents), and as treatment afterradioactive exposure (i.e., radiation mitigators).

In addition, lung cancer is the leading cause of cancer mortality in theUnited States. Despite novel targeted therapeutic agents, improvedstaging and surgical techniques, and increased utilization ofconcomitant chemoradiation therapy for locally advanced lung cancerthere has only been a minimal decrease of overall mortality rates (Tan &Spivack (2009) Lung Cancer 65:129-137). Cancer chemoprevention has beendefined as the use of dietary and pharmacological intervention withspecific natural or synthetic agents designed to prevent, suppress, orreverse the process of carcinogenesis before the development ofmalignancy (Hong & Sporn, (1997). Science 278:1073-1077). One strategyfor lung cancer chemoprevention focuses on the use of agents thatmodulate the metabolism and disposition of tobacco, environmental andother carcinogens through upregulation of detoxifying phase II enzymes.Many synthetic and naturally occurring compounds are known to induce theexpression of phase II enzymes. There have been numerous reports thatsupport the idea that Nrf2/ARE-regulated phase II enzyme induction is ahighly effective strategy for reducing susceptibility to carcinogens. Wehave data to show that flaxseed (FS) and its main lignan SDG, both fromenrichment of the natural material and synthetically derived, areeffective lung cancer chemoprevention agents in a mouse model ofchemical carcinogen-induced lung cancer.

Approximately 85% of lung cancer is caused by smoking. Major lungcarcinogens in tobacco smoke are polycyclic aromatic hydrocarbons,typified by benzo[a]pyrene (BaP). Until better treatments are developed,the best hope for decreasing deaths will be prevention throughscreening, smoking cessation, or chemoprevention. Chemopreventive agentsmust be given for prolonged periods of time in large numbers of exposed,but relatively healthy subjects. They must be safe, non-toxic,palatable, and ideally, affordable. A number of chemopreventive agentshave been studied in lung cancer, however none have met these criteria.One of the most promising approaches is upregulation of Phase IIanti-oxidant and detoxifying enzymes. Unfortunately, the Phase II enzymeactivators tested in patients to date, such as Oltipraz or Sulforophane,have proven to be unacceptably toxic (Pendyala et al. (2001). CancerEpidemiol Biomarkers Prev 10:269-272.). Safe, non-toxic chemopreventiveagents that are effective in preventing the oxidative stress and the DNAdamage induced by lung carcinogens in tobacco smoke are thus desperatelyneeded.

Another well-known environmental carcinogen is asbestos, which refers toa group of naturally occurring hydrated fibrous silicate fibers usedcommercially for insulation. It has now been clearly established in bothanimal models and in patients that asbestos fiber inhalation can lead toneoplastic diseases such as malignant mesothelioma (MM) and lung cancer(Carbone & Yang (2012). Clin Cancer Res 18:598-604; Neri et al. (2012).Anticancer Res 32:1005-1013), as well as pulmonary fibrosis. MM is ahighly aggressive cancer that arises from the mesothelial cells of thepleura and peritoneum with a median survival of about 1 year (Sterman etal. (2005). Clin Cancer Res 11, 7444-7453; Sterman et al. (1999). Chest116, 504-520; Benard et al. (1999). J Nucl Med 40, 1241-1245). Currenttherapies, other than surgery in very early disease, are not curative(Sterman & Albelda (2005). Respirology 10, 266-283.). Presently, MMcauses about 3,000 deaths per year in the US and an additional 5,000deaths/year in Western Europe.

Although asbestos use has been restricted in many western countries, itis still used in many countries around the world and it is estimatedthat more than 2 million tons were mined in 2008 (Survey, B. G. (2010).World Mineral Production 2004-08. Nottingham, UK, British GeologicalSurvey). There will thus likely be a dramatic increase in MM cases inthe third world (especially in India) where the use of asbestos hasincreased with few precautions taken. However, even in the developedworld, important exposures still exist. These include many types ofoccupations that expose workers to pre-existing asbestos (i.e. plumbers,pipefitters, insulators, insulation removal, etc.) as well as superfundasbestos hazardous waste sites. There are also environmental anddomestic exposures. For example, there is an increased risk of MM inareas where mining or asbestos factories have closed.

A major issue in the link between asbestos and cancer is that inhaledasbestos fibers can persist in the lung for very long periods of timeresulting in continuous damage, even if the patient is removed from theexposure. Because of this long latency period (often up to 30-50 years),individuals exposed in the past remain at increased risk of MM and othercancers throughout their lives.

Chemoprevention of cancer aims to prevent, arrest, or reverse either theinitiation phase of carcinogenesis or the progression of neoplasticcells to cancer. Although this definition sounds simple, it has beenvery difficult to find effective chemopreventive agents. First, themechanisms by which carcinogens induce cancer usually involve multiplemechanisms, making efficacy challenging and requiring an agent withmultiple activities. Second, since the agent will be used to prevent asmall number of tumors in a large population of healthy, but at-riskindividuals, it must be extraordinarily non-toxic, well-tolerated, andaffordable.

It is, therefore, also of high importance to identify agents (i.e.,chemopreventive agents) that can be administered before, during, andexposure to carcinogens or other harmful chemical agents, such aschemical warfare agents, chlorine and hypochlorite ions and otherharmful toxicants.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage in a subject inneed thereof, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, degradant or ametabolite thereof. Administration to said subjects encompassesadministration prior to, during and after exposure to damaging radiationexposure. The time prior, during and post could be seconds, minutes,hours, days, weeks, months or even years. The bioactive ingredientencompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for treating orpreventing radiation damage in a subject who has been or will be exposedto radiation, the method comprising: administering to said subject aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolaricirecinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for protectingbiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage resulting fromaccidental radiation exposure in a subject in need thereof, the methodcomprising: administering to said subject an effective amount offlaxseed, its bioactive ingredient, degradant or a metabolite thereof.

In another aspect, the invention relates to a method for protectingbiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage resulting in aging.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue from damage resulting from exposure tochemical carcinogens and toxicants both natural and synthetic.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage resulting fromradiation therapy for cancer treatment in a subject in need thereof, themethod comprising: administering to said subject an effective amount offlaxseed, its bioactive ingredient, degradant or a metabolite thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue from radiation damage in a subject in needthereof, the method comprising: administering to said subject aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolariciresinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof. Insome embodiments, the radiation damage results from accidental radiationexposure. In some embodiments, the radiation damage results fromradiation therapy for cancer (e.g., lung cancer) treatment.

In another aspect, the invention relates to a method for preventingradiation induced damage to a biomolecule (such as genetic material likea nucleic acid, a protein or a lipid), a cell, or a tissue, in a subjectin need thereof, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, or a metabolitethereof.

In another aspect, the invention relates to a method for preventingradiation induced damage to a biomolecule (such as genetic material likea nucleic acid, a protein or a lipid), a cell, or a tissue, in a subjectin need thereof, the method comprising: administering to said subject aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolariciresinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage in a cell, themethod comprising contacting said cell with an effective amount of atleast one bioactive ingredient, wherein said bioactive ingredientcomprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from carcinogen damage in a subject inneed thereof, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, degradant or ametabolite thereof. Administration to said subjects encompassesadministration prior to, during and after exposure to damaging exposureto chemical carcinogens and toxicants both natural and synthetic. Thetime prior, during and post could be seconds, minutes, hours, days,weeks, months or even years. The bioactive ingredient encompassessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule, from carcinogen damage resulting from accidental exposureto chemical carcinogens and toxicants both natural and synthetic in asubject in need thereof, the method comprising: administering to saidsubject an effective amount of flaxseed, its bioactive ingredient,degradant or a metabolite thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from damage from chemical carcinogens andtoxicants both natural and synthetic resulting in lung cancer ormesothelioma.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from carcinogen damage, the methodcomprising: contacting said biomolecule, cell, or tissue with aneffective amount of a bioactive ingredient. Contact with saidbiomolecule, cell, or tissue encompasses contact prior to, during andafter exposure to damaging exposure to chemical carcinogens andtoxicants both natural and synthetic. The time prior, during and postcould be seconds, minutes, hours, days, weeks, months or even years. Thebioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for treating orpreventing carcinogen-induced damage, malignant transformation or cancerdevelopment in subject who has been or will be exposed to one or morecarcinogens from carcinogen-induced cancer, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting asubject exposed to one or more carcinogens from a carcinogen-inducedcancer, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, or a metabolitethereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from damage by hypochlorite ions in asubject in need thereof, the method comprising: administering to saidsubject an effective amount of flaxseed, its bioactive ingredient,degradant or a metabolite thereof. Administration to said subjectsencompasses administration prior to, during and after exposure todamaging exposure to chemical carcinogens and toxicants both natural andsynthetic. The time prior, during and post could be seconds, minutes,hours, days, weeks, months or even years. The bioactive ingredientencompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for treating orpreventing hypochlorite ion-induced damage in a subject who has been orwill be exposed to hypochlorite ions, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from damage by hypochlorite ions, themethod comprising: contacting said biomolecule, cell, or tissue exposedto or to be exposed to hypochlorite ions with an effective amount of abioactive ingredient. Contact with said biomolecule, cell, or tissueencompasses contact prior to, during and after exposure to damagingexposure to chemical carcinogens and toxicants both natural andsynthetic. The time prior, during and post could be seconds, minutes,hours, days, weeks, months or even years. The bioactive ingredientencompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a composition for use in oneof the foregoing methods.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Other features and advantagesof the present invention will become apparent from the followingdetailed description examples and figures. It should be understood,however, that the detailed description and the specific examples whileindicating preferred embodiments of the invention are given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description. It is also contemplated thatwhenever appropriate, any embodiment of the present invention can becombined with one or more other embodiments of the present invention,even though the embodiments are described under different aspects of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effect of increasing doses of γ radiation on Plasmid (pBR322)DNA relaxation. Super coiled (SC) represents the compact form. The opencoiled (OC) form represents the relaxed or the damaged form of theplasmid. (A)—The super coiled (SC) form is the lower prominent band (at3,000 bps) while the open coiled (OC) form is the upper prominent band.Lane 1—1 kb DNA standard ladder, lanes 2 and 3—untreated plasmid DNA,lanes 4 and 5—plasmid DNA exposed to 10 Gy, lanes 6 and 7—plasmid DNAexposed to 25 Gy, and lanes 8 and 9—plasmid DNA exposed to 50 Gy. (B)—SCand OC forms are presented as percent of total plasmid DNA. For eachcondition, all samples were run in duplicates. The data is presented asmean±standard deviation. P<0.05 was considered significant. * and **show the significant difference as compared to untreated SC and OCforms, respectively.

FIG. 2: Effect of increasing concentration of synthetic SDG (S,S), SDG(R,R) and commercial SDG on γ radiation-induced Plasmid (pBR322) DNArelaxation. All samples were exposed to a γ radiation dose of 25 Gray.SDGs concentrations were 25, 50, 100 and 250 μM. In Figures (A), (D),and (G)—representative agarose gel scans of plasmid DNA after exposureto 25 Gy in the presence of 25, 50, 100 and 250 μM SDG (S,S), SDG (R,R)and SDG (commercial) are shown. Lane 1—1 kb DNA standard ladder, lanes 2and 3—untreated plasmid DNA, lanes 4 and 5-25 μM, lanes 6 and 7—50 μM,lanes 8 and 9-100 μM, and lanes 10 and 11-250 μM SDGs. In Figures (B),(E) and (H)—SC and OC forms are presented as percent of total plasmidDNA. For each condition, all samples were run in duplicates. The data ispresented as mean±standard deviation. P<0.05 was consideredsignificant. * and # show the significant difference as compared tountreated SC and OC forms, respectively. ** and ## show significantdifferences as compared to samples exposed to 25 Gy without SDGs. InFigures (C), (F) and (I), SDGs-dependent inhibition of plasmid DNArelaxation is shown. EC50 values were determined from the quadraticequations presented under the curves.

FIG. 3: Effect of increasing doses of γ radiation on calf thymus DNAfragmentation. DNA exposed to γ-radiation generates fragments of smallmolecular weights which move faster than the higher molecular wt. DNA.Determining the density of the low molecular wt DNA fragments (<6,000bps) as compared to the high molecular wt. DNA (>6,000 bps) reflects theextent of radiation-induced damage. (A) Lane 1—1 kb DNA standard ladder,lanes 2 and 3—untreated calf thymus DNA, lanes 4 and 5—DNA exposed to 25Gy, and lanes 6 and 7—DNA exposed to 50 Gy. (B)—High and Low molecularwt DNA forms are presented as percent of total DNA. For each condition,all samples were run in duplicates. The data is presented asmean±standard deviation. P<0.05 was considered significant. * show thesignificant difference as compared to the untreated forms, respectively.

FIG. 4: Effect of increasing concentration of synthetic SDG (S,S), SDG(R,R) and commercial SDG on γ radiation-induced calf thymus DNAfragmentation. All samples were exposed to a γ radiation dose of 50Gray. SDGs concentrations were 25, 50, 100 and 250 μM. In Figures (A),(C), and (E)—representative agarose gel scans of calf thymus DNA afterexposure to 50 Gy in the presence of 25, 50, 100 and 250 μM SDG (S,S),SDG (R,R) and SDG (commercial) are shown. Lane 1—1 kb DNA standardladder, lanes 2 and 3—untreated DNA, lanes 4 and 5-25 μM, lanes 6 andγ-50 μM, lanes 8 and 9-100 μM, and lanes 10 and 11-250 μM SDGs. InFigures (B), (D) and (F)—High and Low molecular wt DNA forms arepresented as percent of total DNA. For each condition, all samples wererun in duplicates. The data is presented as mean±standard deviation.P<0.05 was considered significant. * shows the significant difference ascompared to untreated DNA. # shows the significant difference ascompared to samples exposed to 50 Gy without SDGs.

FIG. 5: Effect of very low concentrations of synthetic SDG (S, S), SDG(R,R) and commercial SDG on γ radiation-induced calf thymus DNAfragmentation. All samples were exposed to a γ radiation dose of 50 Gy.SDGs concentrations were 0.5, 1.0, 5.0 and 10 μM. In Figures (A), (C),and (E)—representative agarose gel scans of calf thymus DNA afterexposure to 50 Gy in the presence of 0.5, 1.0, 5.0 and 10 μM SDG (S,S),SDG (R,R) and SDG (commercial) are shown. Lane 1—1 kb DNA standardladder, lanes 2 and 3—untreated DNA, lanes 4 and 5—0.5 μM, lanes 6 and7—1.0 μM, lanes 8 and 9-5.0 μM, and lanes 10 and 11-10 μM SDGs. InFigures (B), (D) and (F)—High and Low molecular wt DNA forms arepresented as percent of total DNA. For each condition, all samples wererun in duplicates. The data is presented as mean±standard deviation.P<0.05 was considered significant. * shows the significant difference ascompared to untreated DNA. # shows the significant difference ascompared to samples exposed to 50 Gy without SDGs.

FIG. 6: Effect of SDG, SECO, ED and EL on γ radiation-induced calfthymus DNA fragmentation. All samples were exposed to a γ radiation doseof 50 Gy. SDG, SECO, ED and EL were used at 10 μM concentration. (A)representative agarose gel scans of calf thymus DNA after exposure to 50Gy in the presence of 10 μM SDG, SECO, ED and EL are shown. Lane 1—1 kbDNA standard ladder, lanes 2 and 3—untreated DNA, lanes 4, 5 and 6—IR 50Gy, lanes 7 and 8—SDG, lanes 9 and 10—SECO, lanes 11 and 12—ED, andlanes 13 and 14—EL. (B) High and Low molecular wt DNA forms arepresented as percent of total DNA. For each condition, all samples wererun in duplicates. The data is presented as mean±standard deviation.P<0.05 was considered significant. * shows the significant difference ascompared to untreated DNA. # shows the significant difference ascompared to samples exposed to 50 Gy alone.

FIG. 7: Effect of SDG treatment on radiation dose response of murineprimary lung cells. (A) Epithelial Cells; (B) Endothelial Cells and (C)WT Fibroblasts. Cells were treated with different concentrations of SDGfor 6 h prior to gamma irradiation (0, 2, 4, 6, 8 Gy) and incubated. Allvisible colonies were counted on 12-14th day and surviving fraction wasnormalized against control values. Data is represented as mean±SEM. **p≦0.001 * p≦0.01, #p≦0.05 for irradiated cells VS 50 μM SDG pre-treatedirradiated cells.

FIG. 8: Evaluation of the radiation-induced DNA single strand breaks(SSB) in lung cells using the alkaline comet assay. (A) Kineticsevaluation of DNA damage in 2-Gy gamma irradiated primary lung cells(epithelial, endothelial and WT Fibroblasts); At least 100-150 cellswere counted for each treatment. DNA damage was assessed by calculatingthe “tail moment” for each cell (the product of amount of DNA in tail,times the tail length). * p≦0.001 for non-irradiated controls VS theirrespective irradiated cells; (B) Effect of SDG (50 μM) treatment (0, 2,4, 6 hours prior to irradiation) on irradiated primary lung cells. Datais represented as mean±SEM. * p≦0.001, #p≦0.01 for irradiated cells VSSDG pre-treated irradiated cells. Insert: Representative fluorescencephotomicrograph of primary lung epithelial cells. Cells were pre-treatedwith SDG (50 μM) and exposed to the g-radiation (2 Gy), embedded inagarose, lysed, and electrophoresis was done (0.66 V/cm, 25 min),stained with SYBR green and visualized under fluorescence microscope andthereafter examined for comet tail formation. (A) Control cells, (B) SDG(50 μM), (C) IR (2 Gy) after 30 min of exposure, (D) Cells pre-treatedwith SDG (50 μM, 6 h) and irradiated.

FIG. 9: Fluorescent evaluation of the induction of γ-H2AX foci inirradiated murine primary lung cells viz. epithelial cells, endothelialcells and WT fibroblasts. Cells were treated with SDG (50 μM) for 6 hand gamma-irradiated (2 Gy). At desired time interval, cells were fixedin 4% paraformaldehyde, washed, probed with γ-H2AX antibody and nucleiwas counterstained using DAPI. Cells were visualized under afluorescence microscope. Total cells (blue) γ-H2AX positive cells(green) were counted per field and percentage of γ-H2AX positive cellswas calculated. At least 500 cells were counted for each treatment andexperiment done twice. Data is represented as mean±SEM. *p≦0.05, **p≦0.005 for irradiated cells VS. SDG pre-treated irradiated cells.

FIG. 10: Representative panels of immunofluorescence visualization ofγ-H2AX foci (green) in murine lung epithelial cells. Cells werepre-treated with SDG for 6 h, gamma-irradiated (2 Gy) and incubatedfurther for 30 mM Cells were fixed in 4% paraformaldehyde and probedwith γ-H2AX antibody. DNA was counterstained with DAPI (blue). Imageswere acquired using a fluorescence microscope.

FIG. 11: Flow cytometric (FACS) confirmation of the induction of γ-H2AXfoci in irradiated murine primary lung cells. Primary lung cells viz.epithelial cells (A), endothelial cells (B) and WT Fibroblasts (C) weretreated with SDG (50 μM) for 6 h and gamma-irradiated (2 Gy). At desiredtime interval, cells were processed for FACS analysis. Data wasquantified using Summit software and is represented as mean±SEM.*p≦0.05, ** p≦0.01 for irradiated cells VS SDG pre-treated irradiatedcells.

FIG. 12: Evaluation of the radiation-induced apoptotic death in primarylung cells. Quantitative evaluation of the effect of SDG (50 mM)pre-treatment (6 h) on irradiated primary lung cells viz. epithelialcells (A), endothelial cells (B) and WT Fibroblasts (C). Cells werefixed, stained with DAPI and visualized for morphological analysis underfluorescence microscope. For each treatment, at least 500 cells werecounted from 5 different fields and percentage of apoptotic cells wascalculated. Experiment was done twice. Data is represented as mean±SEM.*p≦0.05, ** p≦0.005 for irradiated cells VS. SDG pre-treated irradiatedcells.

FIG. 13: Evaluation of the effect SDG treatment on regulators ofapoptosis in lung epithelial cells. Murine primary lung cells(epithelial cells) were treated with SDG (50 μM) for 6 hours prior to 2Gy exposure. Cells were harvested at 6, 24, and 48 hourspost-irradiation. Total RNA was isolated from epithelial cells atdesired time interval and evaluated by quantitative real time RT-PCRanalysis for Bax and Bcl-2 gene expression (A and B). Analysis wasperformed in triplicate and gene expression was normalized to 18Sribosomal RNA. Bax and Bcl-2 protein levels were assessed by westernblot analysis; representative images (C) and densitometry analysis withnormalization to β-actin. (D and E). Data is represented as mean±SEM.*p≦0.05, **p≦0.01 for IR-exposed cells compared to either SDG treatedcells or SDG+IR treated cells.

FIG. 14: Effect of SDG on radiation induced increases in levels ofactive caspase-3 and cleaved PARP. Murine primary lung cells (epithelialcells) were treated with SDG (50 μM) for 6 hours prior to 2 Gy exposure.Cells were harvested at 6, 24, and 48 hours post-radiation. Cleavedcaspase-3 and cleaved PARP protein levels were assessed by western blotanalysis. (A) Representative images and (B and C) densitometry analysiswith normalization to β-actin. Analysis was performed in duplicate anddata is represented as mean±SEM. *p≦0.05, **p≦0.01 for IR-exposed cellscompared to SDG treated cells.

FIG. 15: SDG Scavenges Hypochlorite Ions. FIG. 15A shows the ClO⁻dependent increase in APF and HPF fluorescence. FIG. 15B showsscavenging of ClO⁻ by SDG. FIG. 15C shows the scavenging effect ofsynthetic SDG diastereomers SDG(S, S) and SDG (R, R). All samples wererun in duplicates. The data are presented as mean±standard error. P<0.05was considered significant. * shows the significant difference ascompared to untreated control.

FIG. 16: SDG Scavenges γ-Radiation-induced Generation of Hypochlorite.FIGS. 16A and B show γ-radiation-induced increase APF and HPFfluorescence. FIGS. 16C and D show the effect of SDG on generation ofhypochlorite, at increasing doses of radiation in Phosphate bufferedsaline (PBS) with either APF or HPF (see FIG. 15 legend). FIGS. 16E, Fand G show γ-radiation-induced chlorination of Taurine. FIG. 16E showshypochlorite-dependent chlorination of taurine. FIG. 16F shows thetaurine chloramine as absorbance for all experimental conditions. FIG.16G shows the hypochlorite concentration under various conditions as inFIG. 16F. For FIGS. 16A-E, all samples were run in duplicates whereasfor FIGS. 16F and G, all samples were run in quadruplets. The data arepresented as mean±standard error. P<0.05 was considered significant. *shows the significant difference as compared to the untreated controls.

FIG. 17: Hypochlorite-induced Calf thymus DNA Damage. FIGS. 17A and Cshow representative agarose gels scans of calf thymus DNA after exposureto HOCl. FIGS. 17B and D show high and low molecular wt DNA fragments aspercent of total DNA. FIGS. 17E and F show the effect of SDG onhypochlorite-induced damage to plasmid DNA. FIG. 17E shows arepresentative agarose gel of plasmid DNA after exposure to HOCl. FIG.17F shows SC and OC forms presented as percent of total plasmid DNA. ForFIG. 17A, Lane 1—1 kb DNA standard ladder, lanes 2 and 3-untreated DNA,lanes 4 and 5-0.1 mM, lanes 6 and 7—0.2 mM, lanes 8 and 9-0.4 mM, lanes10 and 11-0.5 mM and lanes 12 and 13-0.6 mM ClO—. For FIG. 17C, Lane 1—1kb DNA standard ladder, lanes 2 and 3—untreated DNA, lanes 4 and 0.5 mMHOCl, lanes 6 and 7—0.5 mM HOCl+SDG (com) 1 μM, lanes 8 and 9-0.5 mMHOCl+SDG (S,S) 1 μM, lanes 10-11—0.5 mM HOCl+SDG (R,R) 1 μM, lanes12-13—0.5 mM HOCl+quercetin 1 μM and lanes 14 and 15-0.5 mMHOCl+silibinin 1 μM. For FIG. 17E, Lane 1-1 kb DNA standard ladder,lanes 2 and 3—untreated plasmid DNA, lanes 4 and 4.5 mM HOCl, lanes 6and 7—4.5 mM HOCl+SDG 25 μM. For each condition, all samples were run induplicates. The data are presented as mean±standard error. P<0.05 wasconsidered significant. * and # show the significant difference ascompared to untreated DNA.

FIG. 18: Effect of SDG (Pre- and Post-treatment) on Hypochlorite-inducedModification of 2-Aminopurine (2-AP). FIG. 18A shows the representativespectra for all the conditions. FIG. 18B shows the fluorescence at 374nm under different conditions as in FIG. 18A. FIG. 18C shows the %protection by SDG. For each condition, all samples were run induplicates. The data are presented as mean±standard error. P<0.05 wasconsidered significant. * and # show the significant difference ascompared to untreated 2-AP control and treated, respectively.

FIG. 19: SDG prevents γ-radiation-induced modification of 2-aminopurine(2-AP). FIG. 19A shows the representative spectra for all theconditions. FIG. 19B shows the fluorescence at 374 nm. For eachcondition, all samples were run in duplicates. The data are presented asmean±standard error. P<0.05 was considered significant. * and # show thesignificant difference as compared to untreated 2-AP control andtreated, respectively.

FIG. 20: Proposed mechanism of SDG action in DNA protection fromnucleobase chlorination.

FIG. 21: Mechanism of chemoprevention by flaxseed and its lignans. SDGmitigates lung tumorigenesis by tobacco and other environmentalcarcinogens by inhibiting the multi-step carcinogenesis process. Weprovide evidence indicating that the lignan SDG has chemopreventiveactivity through modulation of the Nrf2-regulated Phase IIdetoxification pathway, and perhaps other mechanisms, in both animalmodels. We further provide data to support that the protective effectsof SDG are mediated by the direct ROS scavenging and/or indirectantioxidant/anti-inflammatory properties, and decrease of carcinogentoxicity and DNA damage.

FIG. 22: SDG decreases oxidative DNA damage induced bybenzo-alpha-pyrene in cells. SDG (10 μM) was added to human epithelialcells (A549) that were exposed to 25 μM of the tobacco and environmentalcarcinogen benzo-alpha-pyrene (BaP) and oxidative damage to DNA wasdetected using mass spectrometry as indicated by the presence of8-oxo-7,8-dihydroguanine (8-oxo-dGuo). SDG decreased DNA damage at 3 and6 hours post carcinogen exposure.

FIG. 23: The carcinogen benzo-alpha-pyrene induced ROS in cells.Exposure of murine epithelial cells to BaP induces damaging reactiveoxygen species (ROS) as detected by a redox-sensitive fluorescence dye.As early as 2 hours, post exposure to the carcinogen, a robust increaseof fluorescence intensity indicates ROS generation in cells.

FIG. 24: SDG prevents ROS generation from carcinogen exposure. Mouseepithelial cells were exposed to 10 or 20 μM BaP and an increasingconcentration of SDG (0, 0.1, 0.5, 1, 5 μM SDG) and ROS was detected 2hours later (as determined appropriate in FIG. 23). SDG scavengedharmful ROS to negligible levels.

FIG. 25: SDG prevents genotoxic stress in human epithelial cells exposedto BaP. Exposure of cells to a potent carcinogen such as BaP, inducesgenotoxic stress as indicated by increased levels of p53 protein. Thisis mitigated dose-dependently by the presence of SDG, at 5, 10, 25 and50 μM concentration.

FIG. 26: SDG prevents oxidative DNA damages in human epithelial cellsexposed to BaP. Exposure of cells to a potent carcinogen such as BaP,induces oxidative DNA damage as indicated by increased levels ofgamma-H2AX, a marker for double-stranded DNA breaks. This is mitigateddose-dependently by the presence of SDG, at 5, 10, 25, 50 and 100 μMconcentration.

FIG. 27: SDG prevents DNA adduct formation in human epithelial cellsexposed to BaP. Exposure of cells to a potent carcinogen such as BaP,induces the formation of DNA adducts. DNA adducts are pieces of DNAcovalently linked to a carcinogen and is directly linked to thedevelopment of malignancy. The DNA adduct levels is decreased by thepresence of SDG or its metabolites ED and EL given alone or incombination.

FIG. 28: Mouse model of chemical carcinogen-induced lung tumors. Mice(A/J strain) are given 4 injections intraperitoneally of the tobacco andenvironmental carcinogen BaP (once weekly) at 1 mg/Kg dose. Mice areinitiated on flaxseed or lignan diet at the time of exposure. Mice areevaluated at various times post exposure to determine tumor burden,mouse weight, and overall health profile.

FIG. 29: Flaxseed decreases tumor burden in mice: Gross pathologicalprofile of murine lungs exposed to carcinogen. Representative clinicalimages of murine lungs several months post BaP exposure and dietaryflaxseed administration.

FIG. 30: Flaxseed decreases tumor burden in mice: Histopathologicalprofile of murine lungs exposed to carcinogen. RepresentativeH&E-strained lung sections of murine lungs several months post BaPexposure and dietary flaxseed administration. Nodules indicated by thearrows from mice fed control diet (top panels) or flaxseed (lowerpanels) appear smaller in the flaxseed-fed mice. Each panel represents adifferent animal.

FIG. 31: Flaxseed decreases tumor burden in mice: Quantitativeassessment of tumor burden. Histological murine lung sections wereevaluated morphologically using image analysis software for overalltumor area (A) and nodule size (B). There was a significant decrease inthe area of the lung occupied by tumor in the mice fed a flaxseed diet(p<0.03). Similarly, there was a trend for smaller tumor nodule size.

FIG. 32: Flaxseed decreases tumor burden in mice: Quantitativeassessment of tumor burden. Histological murine lung sections wereevaluated morphologically using Image analysis software for overallnumber of tumor nodules per lung (A) and % tumor invading the lung (B).There was a trend for less tumor nodules per lung (A) and less tumorinvading with flaxseed supplementation (B)

FIG. 33: Flaxseed supplementation prevents wasting effects from lungcancer induced by BaP. Animal weight was measured longitudinally for 200days post BaP exposure. Mice fed a flaxseed diet, exposed to BaP showedhigher weight than those exposed to BaP on control diet.

FIG. 34: Experimental Scheme for Example 5.

FIG. 35: Mammalian lignan metabolites are detectable in blood 4 daysafter daily ingestion of Flaxseed (FS) and Flaxseed Lignan Component(FLC)-supplemented diets. Specifically, Enterodiol (ED) andEnterolactone (EL) can be detected using liquid chromatography, tandemmass spectrometry (LC/MS/MS). Diets were designed to deliver comparablelignan levels, reflected in the detectable lignan metabolite levels inthe 2 diets.

FIG. 36: Flaxseed (FS) and Flaxseed Lignan Component (FLC) given priorto asbestos exposure, blunted abdominal inflammation induced by ipcrocidolite asbestos injection as evidenced by the numbers ofmacrophages (MF), neutrophils (PMN) and lymphocytes (Ly). Specifically,FS and FLC significantly decreased macrophage influx in the abdomen.*p<0.05

FIG. 37: Mice were initiated on FS and FLC diets and exposed to asbestos24 hours later. Cytokines levels (TNFα and IL-1β) in plasma (B, D) andin abdomen, (A, C) were determined using ELISA 3 days post exposureaccording to the experimental scheme in FIG. 34. Both diets indicated atrend towards preventing secretion of pro-inflammatory cytokines in theabdomen and the systemic circulation induced by asbestos exposure.

FIG. 38: Inflammatory cells also trended lower with the diet (A) whileTNFα (b) and IL-1β (C) cytokine levels induced by 400 and 800 mgcrocidolite asbestos were significantly blunted by FLC added in the diet1 day post asbestos exposure (*p<0.05). *p designates significance ascompared to control diet exposed to asbestos.

FIG. 39: Plasma concentration of SDG and metabolites following oralgavage of variable SDG concentrations in mice.

FIG. 40: Antioxidant enzyme gene expression levels in lung followingoral gavage of variable doses of SDG in mice.

FIG. 41: Kinetics of SDG levels in plasma (A) and lung tissues (B)following oral gavage of 100 mg/Kg SDG in mice and corresponding levelsof AOE gene expression (C).

FIG. 42A-B: Experimental Schemes for Example 7.

FIG. 43: Clinical study design for Example 7.

FIG. 44: Western Blot showing that feeding 10% FS increases HO-1 andNQO-1 in mouse nasal epithelium.

FIG. 45: Kinetics of HO-1 gene expression in human buccal epithelialcells after 40 g FS diet. (*P<0.05 from 0 day).

FIG. 46: Kinetics of urinary IsoP levels in one patient on FS.

FIG. 47: Kinetics of urinary 8-oxo-dGuo levels in Normal and Lungtransplant patients on FS.

FIG. 48: SDG or flaxseed diets are hypothesized to decrease asbestosinduced ROS/inflammation.

FIG. 49: Experimental plan (schematic) of asbestos exposure of cells todetect inflammatory cytokine secretion and nitrosative/oxidative stress:

FIG. 50: SDG blunts asbestos-induced ROS secretion by human mesothelialcells in vitro.

FIG. 51: Evaluation of Asbestos-Induced Oxidative Stress (ROS release)in Culture RAW Macrophages: Asbestos-induced ROS was generated shortlypost asbestos exposure and continued for the duration of the observationperiod.

FIG. 52: SDG given to macrophages several hours post exposure toasbestos decreases oxidative stress.

FIG. 53: SDG given to macrophages several hours post exposure toasbestos decreases nitrosative stress.

FIG. 54: SDG given to macrophages several hours post exposure toasbestos decreases inflammatory cytokine secretion (IL-1β).

FIG. 55: SDG given to macrophages several hours post exposure toasbestos decreases inflammatory cytokine secretion (TNF-α).

FIG. 56: Testing SDG in Asbestos-Induced Mesothelioma using two mousemodels: Using at least 2 models of mice genetically predisposed todevelop mesothelioma after asbestos exposure, we will: Evaluate theacute effects of Flaxseed and SDG on a single dose of asbestos in mice;test whether Flaxseed and SDG inhibits the development of tumors ingenetic models of accelerated, asbestos induced MM.

FIG. 57: FLC Diet Enriched in SDG (35% SDG) Given to MEXTAG Mice Exposedto Asbestos Decreased Inflammation.

FIG. 58: Experimental Plan of asbestos exposure of NF2 mice andflaxseed/SDG lignan formulation evaluation:

FIG. 59: Kinetics of abdominal inflammation in NF2 mice post asbestosexposure: Inflammatory cell influx peaked by 3 days and tapered off by 9days post asbestos exposure. Therefore, 3 days was selected as the timepoint to evaluate inflammation in all subsequent experiments.

FIG. 60: Flaxseed and its SDG-rich lignan component bluntedasbestos-induced inflammation (younger mice): Total white blood cells(A) decreased with FS or FLC addition in the diet, albeit notsignificantly. However, when looking at cell differentials, andmacrophage levels in particular, levels were significantly blunted byboth flaxseed and the SDG-lignan diet (B).

FIG. 61: Flaxseed and its SDG-rich lignan component bluntedasbestos-induced inflammation (older mice): Older mice exposed toabdominal asbestos (A) are more sensitive to asbestos by presenting withapprox. 3,000,000 WBC/mL of abdominal lavage fluid as compared to just300,000 cells/mL (10-fold higher). Results indicated that theinflammatory cells Neutrophils (B) and Macrophages (C) were bothsignificantly higher in older than in younger mice.

FIG. 62: Flaxseed lignan extract enriched in SDG (given in dietformulation) blunts asbestos inflammation in older mice: Male NF2(129SV) (+/−) mice were injected (intraperitoneal) with 400 μg ofasbestos on Day 0. Mice were initiated on the test diets (0% FS or 10%FLC) the week prior to asbestos exposure (Day-7) and sacrificed on Day 3post-asbestos exposure. Abdominal lavage (AL) was performed with 5 mL1×PBS (1 ml of belly lavage fluid was centrifuged and the supernatantwas frozen). Plasma was collected at frozen at −80°. Cells wereevaluated in lavage fluid and showed that total WBC and neutrophils,macrophages and eosinophils were all significantly decreased by theSDG-rich diet.

FIG. 63: Flaxseed lignan extract enriched in SDG (given in dietformulation) blunts asbestos inflammatory cytokine secretion andnitrosative stress in older mice: Male NF2 (129SV)(+/−) mice wereinjected (intraperitoneal) with 400 μg of asbestos on Day 0. Mice wereinitiated on test diets (0% FS or 10% FLC) the week prior to asbestosexposure (Day 7) and sacrificed on Day 3 post-asbestos exposure.Abdominal lavage (AL) was performed with 5 mL 1×PBS (1 mL of bellylavage fluid was centrifuged and the supernatant frozen). Plasma wascollected at frozen at −80°. Levels of cytokines IL1β and TNFα as wellas nitrites were significantly blunted by the SDG-rich diet.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

Provided herein are therapeutic and prophylactics methods of usingflaxseed, its bioactive ingredients, or its metabolites forradioprotection and chemoprevention. In exemplary embodiments, thebioactive ingredient comprises secoisolariciresinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, isomers (including stereoisomers) thereof, or acombination thereof.

The inventors of this application have found that flaxseed, itsbioactive ingredients, and/or its degradants or metabolites areeffective in protecting biomolecules, cells, and tissues from radiationdamage, hypochlorite ion-induced damage, carcinogen-induced damage andmalignancy. Accordingly, the inventors have found that flaxseed, itsbioactive ingredients, or its metabolites can be used for protectingbiomolecules, cells, and tissues from radiation damage, hypochloriteion-induced damage, carcinogen damage and cancer development.

Subjects in need of radioprotection or radiation mitigation according tomethods provided herein are subjects who will, are, or have been exposedto potentially deleterious amounts of radiation. It will be understoodthat such exposure may be a single exposure, periodic exposure, sporadicexposure or ongoing exposure to the radiation. It is also understoodthat such radiation exposure includes accidental exposure, incidental orintentional exposure.

Examples of subjects who may be in need of radioprotection or radiationmitigation according to the methods of the present invention include butare not limited to, patients who are exposed to radiation as part oftherapeutic regimen (e.g., cancer patients who require radiationtherapy), subjects who are exposed to radiation for to diagnose adisease or condition (e.g., subjects receiving dental or bone X-rays,patients receiving PET scans, CT scans and the like). Examples ofsubjects who may be in need of radioprotection or radiation mitigationaccording to the methods of the present invention also include those whomay be exposed to radiation as a result of their profession or lifestyle choices (e.g., airplane flight crews or other frequent airtravelers, and even space travelers, who are exposed to higher thanaverage radiation levels; laboratory technicians and other workers; orthose exposed through the use of electronic devices) or those exposed toaccumulations of radon (e.g., accumulations in dwellings or mines) oroutdoor workers or sunbathers exposed to natural radiation from the sun.Other subjects who may be in need of radioprotection according to themethods of the present invention include those who are accidentallyexposed to radiation, such as leaks or spills, (e.g., nuclear reactorleaks or accidents or laboratory spills). Also contemplated are thoseexposed to radiation as a result of the detonation of a nuclear warhead,as a result of war or terrorism. Additional subjects encompassed arethose who are exposed to a terrorist's detonation of conventionalexplosives that disperse radioactive materials.

Subjects in need of chemoprevention according to methods provided hereinare subjects who will, are, or have been exposed to potentiallydeleterious amounts of carcinogens or other toxicants. It will beunderstood that such exposure may be a single exposure, periodicexposure, sporadic exposure or ongoing exposure to one or combination ofseveral synthetic or naturally occurring carcinogens or other toxicants,such as chemical warfare agents. It is also understood that suchexposure includes accidental exposure, incidental or intentionalexposure. It will also be understood that such exposure may be directexposure or indirect exposure. For example, indirect exposure tohypochlorite ions may be the result of direct exposure to ionizingradiation.

Examples of subjects who may be in need of chemoprevention according tothe methods of the present invention include but are not limited tothose who may be exposed to carcinogens or other toxicants as a resultof their profession or life style choices (e.g., workers in the oilindustry; toll booth attendants exposed to automobile exhaust particles;laboratory technicians and other workers). Other subjects who may be inneed of chemoprevention according to the methods of the presentinvention include those who are accidentally exposed to carcinogens,such as leaks or spills of carcinogens in the drinking water or the air(asbestos, polyaromatic hydrocarbons). Also contemplated are thoseexposed to carcinogens as a result of a habit (smokers). Additionalsubjects encompassed are those who are exposed to a terrorist's act todisperse carcinogen and other cancer promoting materials, such aschemical warfare agents.

In one aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage in a subject inneed thereof, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, degradant or ametabolite thereof. Administration to said subjects encompassesadministration prior to, during and after exposure to damaging radiationexposure. The time prior, during and post could be seconds, minutes,hours, days, weeks, months or even years. The bioactive ingredientencompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for treating orpreventing radiation damage in a subject who has been or will be exposedto radiation, the method comprising: administering to said subject aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolaricirecinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for protectingbiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage resulting fromaccidental radiation exposure in a subject in need thereof, the methodcomprising: administering to said subject an effective amount offlaxseed, its bioactive ingredient, degradant or a metabolite thereof.

In another aspect, the invention relates to a method for protectingbiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage resulting in aging.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue from damage resulting from exposure tochemical carcinogens and toxicants, including chemical warfare agents,both natural and synthetic.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage resulting fromradiation therapy for cancer treatment in a subject in need thereof, themethod comprising: administering to said subject an effective amount offlaxseed, its bioactive ingredient, degradant or a metabolite thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue from radiation damage in a subject in needthereof, the method comprising: administering to said subject aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolariciresinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof. Insome embodiments, the radiation damage results from accidental radiationexposure. In some embodiments, the radiation damage results fromradiation therapy for cancer (e.g., lung cancer) treatment.

In another aspect, the invention relates to a method for preventingradiation induced damage to a biomolecule (such as genetic material likea nucleic acid, a protein or a lipid), a cell, or a tissue, in a subjectin need thereof, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, or a metabolitethereof.

In another aspect, the invention relates to a method for preventingradiation induced damage to a biomolecule (such as genetic material likea nucleic acid, a protein or a lipid), a cell, or a tissue, in a subjectin need thereof, the method comprising: administering to said subject aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolariciresinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from radiation damage in a cell, themethod comprising contacting said cell with an effective amount of atleast one bioactive ingredient, wherein said bioactive ingredientcomprises secoisolariciresinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from carcinogen damage in a subject inneed thereof, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, degradant or ametabolite thereof. Administration to said subjects encompassesadministration prior to, during and after exposure to damaging exposureto chemical carcinogens and toxicants both natural and synthetic. Thetime prior, during and post could be seconds, minutes, hours, days,weeks, months or even years. The bioactive ingredient encompassessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule, from carcinogen damage resulting from accidental exposureto chemical carcinogens and toxicants both natural and synthetic in asubject in need thereof, the method comprising: administering to saidsubject an effective amount of flaxseed, its bioactive ingredient,degradant or a metabolite thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from damage from chemical carcinogens andtoxicants both natural and synthetic resulting in lung cancer ormesothelioma.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from carcinogen damage, the methodcomprising: contacting said biomolecule, cell, or tissue with aneffective amount of a bioactive ingredient. Contact with saidbiomolecule, cell, or tissue encompasses contact prior to, during andafter exposure to damaging exposure to chemical carcinogens andtoxicants both natural and synthetic. The time prior, during and postcould be seconds, minutes, hours, days, weeks, months or even years. Thebioactive ingredient encompasses secoisolaricirecinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for treating orpreventing carcinogen-induced damage, malignant transformation or cancerdevelopment in subject who has been or will be exposed to one or morecarcinogens from carcinogen-induced cancer, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting asubject exposed to one or more carcinogens from a carcinogen-inducedcancer, the method comprising: administering to said subject aneffective amount of flaxseed, its bioactive ingredient, or a metabolitethereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from damage by hypochlorite ions in asubject in need thereof, the method comprising: administering to saidsubject an effective amount of flaxseed, its bioactive ingredient,degradant or a metabolite thereof. Administration to said subjectsencompasses administration prior to, during and after exposure todamaging exposure to chemical carcinogens and toxicants both natural andsynthetic. The time prior, during and post could be seconds, minutes,hours, days, weeks, months or even years. The bioactive ingredientencompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a method for treating orpreventing hypochlorite ion-induced damage in a subject who has been orwill be exposed to hypochlorite ions, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.

In another aspect, the invention relates to a method for protecting abiomolecule (such as genetic material like a nucleic acid, a protein ora lipid), a cell, or a tissue, from damage by hypochlorite ions, themethod comprising: contacting said biomolecule, cell, or tissue exposedto or to be exposed to hypochlorite ions with an effective amount of abioactive ingredient. Contact with said biomolecule, cell, or tissueencompasses contact prior to, during and after exposure to damagingexposure to chemical carcinogens and toxicants both natural andsynthetic. The time prior, during and post could be seconds, minutes,hours, days, weeks, months or even years. The bioactive ingredientencompasses secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.

In another aspect, the invention relates to a composition for use in oneof the foregoing methods.

Flaxseed, its bioactive ingredients, and its metabolites are known inthe art and described in U.S. Patent Publication Nos. 2010/0239696;2011/0300247; and 2014/0308379; and in International Patent PublicationNo. WO2014/200964, each of which is incorporated by reference herein inits entirety.

The primary lignan found in flaxseed is 2,3-bis(3-methoxy-4-hydroxybenzyl) butane-1,4-diol (secoisolariciresinol orSECO), which is stored as the conjugate secoisolariciresinol diglucoside(SDG) in its native state in the plant. SDG is metabolized in the humanintestine to enterodiol (ED), and enterolactone (EL). Synthetic analogsof enterodiol and enterolactone are known (see, e.g., Eklund et al.,Org. Lett., 2003, 5:491).

A “degradant” is a product of the breakdown of a molecule, such as SDG,into smaller molecules.

A “metabolite” is a substance produced by metabolism or by a metabolicprocess. For example, a metabolite of SDG is EL or ED.

It will be appreciated by one skilled in the art that a metabolite maybe a chemically synthesized equivalent of a natural metabolite.

An “analog” is a compound whose structure is related to that of anothercompound. The analog may be a synthetic analog.

An “ingredient” or “component” is an element or a constituent in amixture or compound.

A “product” is a substance resulting from a chemical reaction.

An “extract” is a preparation containing an active principle orconcentrated essence of a material, for example, from flaxseed.

“Pharmaceutical composition” refers to an effective amount of an activeingredient, e.g., (S,S)-SDG (R,R)-SDG, meso-SDG, SDG, SECO, EL, ED andanalogs thereof, together with a pharmaceutically acceptable carrier ordiluent. A “therapeutically effective amount” refers to that amountwhich provides a therapeutic effect for a given condition andadministration regimen.

The compositions described herein may include a “therapeuticallyeffective amount.” A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the composition toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which toxic or detrimental effects of the moleculeare outweighed by the therapeutically beneficial effects.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused herein includes both one and more than one such excipient.

The pharmaceutical compositions can be administered to a subject by anysuitable method known to a person skilled in the art, such as orally,parenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, subcutaneously, intra-peritonealy,intra-ventricularly, intra-cranially, intra-vaginally, intratumorally,or bucally. Controlled release may also be used by embedding the activeingredient in an appropriate polymer which may then be insertedsubcutaneously, intratumorally, bucally, as a patch on the skin, orvaginally. Coating a medical device with the active ingredient is alsocovered.

In some embodiments, the pharmaceutical compositions are administeredorally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Suitable solidoral formulations include tablets, capsules, pills, granules, pelletsand the like. Suitable liquid oral formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In someembodiments, the active ingredient is formulated in a capsule. Inaccordance with this embodiment, the compositions of the presentinvention comprise, in addition to the active compound and the inertcarrier or diluent, drying agent, in addition to other excipients aswell as a gelatin capsule.

In some embodiments, the pharmaceutical compositions are administered byintravenous, intra-arterial, or intra-muscular injection of a liquidpreparation. In some embodiments, the pharmaceutical composition is aliquid preparation formulated for oral administration. In someembodiments, the pharmaceutical composition is a liquid preparationformulated for intravaginal administration. Suitable liquid formulationsinclude solutions, suspensions, dispersions, emulsions, oils and thelike. In some embodiments, the pharmaceutical compositions areadministered intravenously and are thus formulated in a form suitablefor intravenous administration. In another embodiment, thepharmaceutical compositions are administered intra-arterially and arethus formulated in a form suitable for intra-arterial administration. Insome embodiments, the pharmaceutical compositions are administeredintra-muscularly and are thus formulated in a form suitable forintra-muscular administration. In some embodiments, the pharmaceuticalcompositions are administered intra-bucally and are thus formulated in aform suitable for buccal administration.

In some embodiments, the pharmaceutical compositions are administeredtopically to body surfaces and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops, controlled release polymers and thelike. For topical administration, the flaxseed, its bioactiveingredient, or a metabolite thereof is prepared and applied as asolution, suspension, or emulsion in a physiologically acceptablediluent with or without a pharmaceutical carrier.

In some embodiments, the pharmaceutical compositions provided herein arecontrolled-release compositions, i.e. compositions in which theflaxseed, its bioactive ingredient, or a metabolite thereof is releasedover a period of time after administration. Controlled- orsustained-release compositions include formulation in lipophilic depots(e.g. fatty acids, waxes, oils). In other embodiments, the compositionis an immediate-release composition, i.e. a composition in which all theflaxseed, its bioactive ingredient, or a metabolite thereof is releasedimmediately after administration.

In some embodiments, compositions for use in the methods provided hereinare administered at a therapeutic dose once per day. In someembodiments, the compositions are administered once every two days,twice a week, once a week, or once every two weeks.

Techniques for extracting and purifying SDG are known in the art anddescribed in U.S. Pat. No. 5,705,618, which is incorporated herein byreference. Techniques for synthesizing SDG, its stereoisomers andanalogs are described in Mishra O P, et al. Bioorganic & MedicinalChemistry Letters 2013, (19):5325-5328 and in International PatentPublication No. WO2014/200964, which are hereby incorporated byreference in their entireties. Bioactive components for use in themethods provided herein may also be chemically synthesized directly intothe mammalian, readily metabolizable forms, Enterodiol (ED) orEnterolactone (EL), as is known in the art.

(S,S)-SDG (R,R)-SDG, (S,R)-SDG (R,S)-SDG, meso-SDG, SECO, EL, ED or ananalog thereof may be administered at a dose of 0.1 ng/kg to 500 mg/kg.

The treatment with (S,S)-SDG (R,R)-SDG, (S,R)-SDG (R,S)-SDG, meso-SDG,SDG, SECO, EL, ED or an analog thereof is a single administration toseveral days, months, years, or indefinitely.

As used herein, “treating” may refer to either therapeutic treatment orprophylactic or preventative measures, wherein the object is to preventor lessen the targeted pathologic condition or disorder as describedherein, or both. Therefore, compositions for use in the methods providedherein may be administered to a subject before the exposure, e.g., toradiation, a carcinogen, a toxicant, or hypochlorite ions. In somecases, compositions for use in the methods provided herein may beadministered to a subject after the exposure. Thus treating a conditionas described herein may refer to preventing, inhibiting, or suppressingthe condition in a subject.

Furthermore, as used herein, the terms “treat” and “treatment” refer totherapeutic treatment, as well prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological change associated with a disease or condition. Beneficialor desired clinical results include, but are not limited to, alleviationof symptoms, diminishment of the extent of a disease or condition,stabilization of a disease or condition (i.e., where the disease orcondition does not worsen), delay or slowing of the progression of adisease or condition, amelioration or palliation of the disease orcondition, and remission (whether partial or total) of the disease orcondition, whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already having beenexposed, e.g., to radiation, a carcinogen, a toxicant, or hypochloriteions, as well as those prone to being exposed or those expecting to beexposed.

In some embodiments, subjects in need of treatment and the methods andcompositions described herein may include, but are not limited to,subjects with lung diseases and disorders, such as asthma, cancer, COPD,and mesothelioma. In some embodiments, suitable subjects may includesubjects with disorders and conditions associated with aging, such ascardiovascular disorders and conditions, sagging skin and centralnervous system (CNS) diseases (e.g., Alzheimer's dementia). In someembodiments, suitable subjects may include skin disorders and conditions(e.g., psoriasis), as well as subjects with cosmetic skin conditions(e.g., wrinkles and age spots). In some embodiments, suitable subjectsmay include subjects with gastrointestinal disorders and conditions,such as IBD and chron's disease. In some embodiments, suitable subjectsmay include subjects with cardiovascular disorders and conditions. Insome embodiments, suitable subjects may include subjects with melanoma.In some embodiments, suitable subjects may include subjects with oculardiseases, such as macular degeneration. In some embodiments, suitablesubjects may include subjects with cancer, such as breast cancer,prostate cancer and uterine cancer. In some embodiments, suitablesubjects include subjects with cognitive impairment and other cognitivedisorders.

The term “subject” includes mammals, e.g., humans, companion animals(e.g., dogs, cats, birds, and the like), farm animals (e.g., cows,sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g.,rats, mice, guinea pigs, birds, and the like). In addition to humans,the subject may include dogs, cats, pigs, cows, sheep, goats, horses,buffalo, ostriches, guinea pigs, rats, mice, birds (e.g., parakeets) andother wild, domesticated or commercially useful animals (e.g., chicken,geese, turkeys, fish). The term “subject” does not exclude an individualthat is normal in all respects. The term “subject” includes, but is notlimited to, a human in need of therapy for, or susceptible to, acondition or its sequelae.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Synthetic (S,S) and (R,R)-SecoisolariciresinolDiglucosides (SDGs) Protect Naked Plasmid and Genomic DNA from GammaRadiation Damage

As a consequence of nuclear disintegration, three types of radiationsare produced namely, the alpha (a) with a positive charge, the beta (β)with a negative charge and the gamma (γ) with no charge. In case ofγ-radiation, the electromagnetic wave has a very small wavelength(<0.005 nm) and thus has high energy which is capable of ionizingmolecules and atoms. In biological systems or in solution, ionizingradiation generates hydroxyl radicals (.OH) by water radiolysis. Thesehydroxyl radicals (.OH) are the predominant source of ionizingradiation-induced damage to cellular components including lipids,proteins and genomic DNA. The hydroxyl radicals (.OH) produced byγ-radiation result in single-strand and double-strand breaks in DNA. The(.OH) radicals damage DNA by abstracting H-atoms from the deoxyriboseand purine as well as pyrimidine bases or add to the double bonds of thebases. These reactions result in DNA strand breaks.

Compounds with antioxidant and free radical scavenging properties canfunction as radioprotectors and prevent radiation-induced DNA damage.Due to complex extraction, purification and enrichment methods toisolate secoisolariciresinol diglucoside (SDG) from natural resourcesassociated with high costs, variability and difficulty to produce largequantities of SDG to make preclinical and clinical testing feasible, SDGwas chemically synthesized. Using the natural compounds vanillin andglucose, two enantiomers (their structures are depicted below) of SDG:SDG (S,S) and SDG (R,R), were successfully synthesized (Mishra et al.,Bioorganic & Medicinal Chemistry Letters 2013, (19):5325).

SDG has been shown in many studies by Christofidou-Solomidou et al., inaddition to others, to be a potent antioxidant agent and a potent freeradical scavenger. Importantly, in a recent study, the synthetic SDGenantiomers have been shown to possess strong antioxidant and freeradical, scavenging characteristics (Mishra et al., Bioorganic &Medicinal Chemistry Letters 2013, (19):5325-5328). In the presentexample, the radioprotective properties of the synthesized SDGenantiomers (S,S)-SDG and (R,R)-SDG as they compare to the commercialSDG were investigated and evaluated. The radioprotective characteristicsof the three compounds were assessed using the plasmid DNA relaxationassay by determining the ability of the SDGs to prevent the super coilto open coil plasmid DNA (pBR322) conversion following the exposure ofthe plasmid to γ-irradiation as well as by evaluating inhibition ofgenomic DNA fragmentation following the exposure of the DNA toγ-irradiation. SDG is metabolized by intestinal bacteria to producesecoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL).Therefore, the effect of these metabolites of SDG onγ-irradiation-induced fragmentation of genomic DNA was also evaluated.

Material and Methods Chemicals

Plasmid DNA (pBR322), ethidium bromide, ultrapure 10×TAE buffer and 1 kbplus DNA ladder were purchased from Invitrogen (Life Technologies,Carlsbad, Calif.). Agarose (ultrapure) and calf thymus DNA werepurchased from Sigma-Aldrich (St. Louis, Mo.). SecoisolariciresinolDiglucoside (SDG) (commercial), Secoisolariciresinol (SECO), enterodiol(ED) and enterolactone (EL) were purchased from Chromadex (Irvine,Calif.).

Synthesis of Secoisolariciresinol Diglucoside (SDG)

Synthetic SDG (R,R) and SDG (S,S) stereoisomers were synthesized asdescribe in Mishra et al., Bioorganic & Medicinal Chemistry Letters2013, (19):5325-5328.

Exposure of Plasmid DNA and Calf Thymus DNA to γ-Radiation

Plasmid DNA (pBR322) or calf thymus DNA samples with or without varyingconcentrations of SDG (R,R), SDG (S,S) and SDG (commercial) were exposedto γ-radiation with a Mark 1 cesium (Cs-137) irradiator (J. L. Shepherd,San Fernando, Calif.) at a dose rate of 1.7 Gy/min in phosphate bufferedsaline, pH 7.4 (PBS).

Determination of Radiation-Induced Plasmid DNA Relaxation

The effect of test compounds on radiation-induced strand breaks andsupercoil (SC) to open coil (OC) conversion was determined using plasmidDNA (pBR322) (Life Technologies, Carlsbad, Calif.). Plasmid DNA (500 ng)in PBS (pH 7.4) was mixed with various concentrations (25-250 μM) of SDG(R,R), SDG (S,S) and SDG (commercial) and irradiated at 25 Gy in PBS. At30 min post radiation exposure, samples were mixed with loading dye andsubjected to agarose (1%) gel electrophoresis in TAE buffer (pH, 8.3) at100 V. The gel was stained with ethidium bromide (0.5 μg/ml) for 40 min,washed for 20 min and then visualized on a UV trans-illuminator(Bio-Rad, Hercules, Calif.). The captured gel images were scanned andthe density of the open coiled (OC) and super coiled (SC) plasmid DNAbands determined by Gel-doc image analyzer program. The density of theSC and OC plasmid DNA was expressed as % of the total density (OC+SC).

Determination of Radiation-Induced DNA Fragmentation

The effect of test compounds on radiation-induced strand breaks in DNAwas determined using calf thymus DNA (Sigma, St. Louis, Mo.). DNA (500ng) in PBS (pH 7.4) was mixed with varying concentrations (25-250 μM) ofSDG (R,R), SDG (S,S) and SDG (commercial) and irradiated at 50 Gy for 30min. A second series of experiments were performed at varyingconcentrations ranging from 0.5-10 μM. Samples were mixed with loadingdye and subjected to agarose (1%) gel electrophoresis in TAE buffer (pH,8.3) at 100 V. The gel was stained with ethidium bromide (0.5 μg/ml) for40 min, washed for 20 min and then visualized on a UV trans-illuminator(Bio-Rad, Hercules, Calif.). The captured gel images were scanned andthe density of the calf thymus DNA fragments was determined by Gel-Proimage analyzer program (Media Cybernetics, Silver Spring, Md.). Thedensity of the low mol. wt (<6,000 bps) and the high mol. wt (>6,000bps) fragments of calf thymus DNA was expressed as % of the totaldensity (low mol wt.+high mol. wt.).

Data Analysis

Data obtained are presented as mean values±standard deviation. The datawere subjected to one-way analysis of variance (ANOVA) with post-hoccomparison using Bonferroni correction using Statview Program. P value≦0.05 was considered as significant.

Results

The radioprotective potential of synthetic SDG (R,R), SDG (S,S) and SDG(commercial) was determined using plasmid DNA (pBR322). Theradioprotection assay used in this study is based on the principle thatplasmid DNA following exposure to γ-radiation moves slower than theunexposed plasmid DNA. This is simply due to the fact that the supercoiled plasmid DNA moves faster in the agarose gel due to its compactsize. By comparison, the radiation-induced nicks in the plasmid DNAunravel super coil resulting in a relatively lager size plasmid whichmoves with a slower rate in the gel. Therefore, determining the densityof the open coiled as compared to super coiled plasmid DNA reflects theextent of radiation-induced damage.

Radiation causes a dose-dependent SC to OC DNA plasmid conversion

To select a radiation dose that causes significant DNA damage yet allowsfor a therapeutic window to test a radiation mitigating agent, plasmidDNA was exposed to 10, 25 and 50 Gy gamma radiation. The resultspresented in FIG. 1A show that there is a radiation dose-dependentincrease in OC form as well as a radiation dose-dependent decrease in SCform of the plasmid DNA. The distribution of SC and OC (FIG. 1B) showsthat the % of SC decreased from 68.73±2.54% to 50.91±2.31, 38.37±3.73and 35.66±4.24% (p<0.05), in 0, 10, 25 and 50 Gy, respectively. At thesame time, the % of OC increased from 31.26±2.50% to 49.08±2.31%61.62±3.73% and 67.33±4.24% (p<0.05), in 0, 10, 25 and 50 Gy,respectively. Based on these initial experiments, a radiation dose of 25Gy (at which a considerable and clearly demonstrable damage wasachieved) was selected for the subsequent experiments to determine theradioprotective characteristics of the different SDGs.

Radioprotective Activity of Synthetic SDG Using Plasmid DNA RelaxationAssay

Plasmid DNA was exposed to the selected dose of 25 Gy gamma radiation(see FIG. 1) and the % inhibition of DNA damage (SC to OC formation) wasdetermined for each of the SDG agents (synthetic and commercial) atvarious concentrations (25-2501.1M).

A representative gel blot of plasmid DNA after exposure to 25 Gy in thepresence of 25, 50, 100 and 250 μM SDG (S,S) is shown in FIG. 2A andsemiquantitative densitometric analysis is shown in FIG. 2B while %inhibition as compared to control is shown in FIG. 2C. Interestingly, inthe presence of increasing concentrations of SDG (S,S) (25, 50, 100 and250 μM), the proportion of the SC form increased and the density of OCform decreased significantly (p<0.05) and dose-dependently. Using the %inhibition plot (FIG. 2C), the EC₅₀ value can be determined for eachagent (i.e, the effective concentration (EC) needed to prevent 50% ofplasmid relaxation at 25 Gy) which for SDG (S,S) is 141.77 μM. Thisvalue for preventing plasmid DNA relaxation is comparable to the EC₅₀value for scavenging DPPH free radicals. These results demonstrate theradioprotective characteristics of the synthetic SDG (S,S) enantiomer.Similar results were shown for the SDG (R,R) enantiomer (Figure, 2D-F)and SDG (commercial) (FIG. 2, G-I) with an EC₅₀ of 127.96 μM and 98.38μM, respectively. These values for preventing plasmid DNA relaxation arecomparable to the respective EC50 value for scavenging DPPH freeradicals. These results demonstrate the radioprotective characteristicsof both synthetic and commercially available, natural SDG.

Radiation Causes Dose-Dependent DNA Fragmentation from High to LowMolecular Weight Fragments

Radiation induces an increase in DNA fragmentation as shown in the DNAgel in FIG. 3A. Based on size, the calf thymus DNA fragments weredivided into two groups: high mol. wt. (>6,000 bps) size and low mol.wt. (<6,000 bps) size. The distribution (FIG. 3B) of high and the lowmol. wt. fragments show that the % of the high mol. wt. DNA decreasedfrom 88.16±0.50% to 67.82±7.89 and 34.94±4.45% (p<0.05) at 25 and 50 Gy,respectively. At the same time, the proportion of low mol. wt. fragmentsincreased from 11.83±0.50 to 32.17±7.89% and 65.05±4.45% (p<0.05) at 25and 50 Gy, respectively. Results (FIG. 3B) show a significant decreasein high mol. wt. DNA and a significant increase in low mol. wt. DNAfragments indicating damage to DNA at 50 Gy. Based on these initialexperiments, a radiation dose of 50 Gy (at which a clear demonstrablecalf thymus DNA fragmentation was observed), was selected for thefollowing experiments determining the radioprotection characteristic ofdifferent SDGs.

Radioprotective Activity of Synthetic SDG Using Calf Thymus DNAFragmentation Assay

The radioprotective potential of synthetic SDG (R,R), SDG (S,S) and SDG(commercial) was determined using radiation-induced fragmentation ofcalf thymus DNA as described above.

High SDG concentration (25-250 μM):

FIG. 4A shows a representative DNA gel of calf thymus DNA after exposureto 50 Gy in the presence of 25, 50, 100 and 250 μM SDG (S,S). In thepresence of increasing concentrations of SDG (S,S) (25, 50, 100 and 250μM), the proportion of the high mol. wt. DNA form increasedsignificantly (p<0.05) following radiation exposure while the low mol.wt. fragments decreased. The distribution of high and low mol. wt sizeDNA forms in presence of various concentrations of SDG (S,S) ispresented in FIG. 4B. These results demonstrate the radioprotectivecharacteristic of our synthetic SDG (S,S) enantiomer using calf thymusgenomic DNA. Similarly, results presented in FIGS. 4C-D and 4E-F showthe radioprotective properties of synthetic SDG (R,R) and SDG(commercial), respectively. These results demonstrate theradioprotective characteristic of synthetic SDG (R,R) and (S,S)enantiomers using calf thymus genomic DNA.

To further determine the lower limits of SDG in DNA protection, a seriesof DNA fragmentation experiments testing lower concentrations of all 3SDGs were performed, ranging from 0.5-10 μM.

Low SDG concentration (0.5-10 μM):

The results of experiments performed at low concentrations of SDG (S,S),SDG (R,R) and SDG (commercial) as compared to their EC₅₀ values forantioxidant and free radical scavenging activity are presented in FIG.5. Similarly to the higher SDG concentrations, these results presentedin this section using calf thymus DNA fragmentation assay demonstratethat the synthetic SDG (S,S), and SDG (R,R) enantiomers possess strongradioprotection characteristics even at low concentrations.

Radioprotective Activity of SDG Metabolites Using Calf Thymus DNAFragmentation Assay

The radioprotective potential of SDG metabolites SECO, ED and EL wasdetermined and compared with SDG using radiation-induced fragmentationof calf thymus DNA as described above. The concentration of 10 μM ofeach the test agent was selected based on previous findings shown aboveas a median effective dose. The results are shown in FIG. 6. The datademonstrate that SDG and its metabolites SECO, ED, EL are equipotentwith respect to their radioprotective properties.

Discussion

The results of the present example show that synthetic SDG (S,S) and SDG(R,R) enantiomers possess strong radioprotective properties. Theradioprotection potential of these enantiomers, as determined usingplasmid DNA (pBR322), increased with increases in their concentration.These synthetic SDG (S,S) and SDG (R,R) enantiomers prevented theradiation-induced damage to plasmid DNA in a concentration-dependentmanner. The radioprotection potential of the synthetic isomers of SDGwas comparable to commercial SDG. Synthetic enantiomers SDG (S,S) andSDG (R,R) also prevented the radiation-induced DNA fragmentation of calfthymus genomic DNA. At the lowest concentration tested, SDG (S,S) andSDG (R,R) completely prevented the radiation induced generation of lowmol. wt. fragments of calf thymus DNA demonstrating strongradioprotective characteristics of synthetic SDG (S,S) and SDG (R,R)enantiomers. Results using low concentrations of SDG (S,S), SDG (R,R)and SDG (commercial) indicated that the concentration required forprotecting calf thymus DNA from γ radiation damage is much lower ascompared to the EC₅₀ values for their antioxidant and free radicalscavenging activity. Importantly, the mammalian lignan metabolites ofSDG, SECO, ED and EL showed equally potent DNA-protective properties.

Flavonoids possess strong antioxidant activity; specifically, suchpolyphenols possess free radical-scavenging activities, and are known tobe more effective antioxidants in vitro than vitamins E and C. Dietaryand medicinal plants possessing antioxidant properties are also known toprevent many human diseases associated with oxidative stress and areuseful radioprotectors. Antioxidants, including vitamins and minerals,suppressed the levels of clastogenic factors in Chernobyl workers manyyears after radiation exposure. We have been investigating the role ofwhole grain dietary flaxseed, a grain rich in lignan polyphenols, aswell as of flaxseed lignan formulations enriched in SDG, inradiation-induced damage using a mouse model of thoracic radiationdamage. We have shown that flaxseed ameliorated the radiation-inducedinflammation and oxidative stress in mice when administered both priorto and after radiation exposure. We also demonstrated that irradiatedmice fed diets containing only the lignan component of flaxseed,enriched in the lignan biphenol SDG, also showed significantly improvedhemodynamic measurements and survival while also improving lunginflammation and oxidative tissue damage. These studies indicated thatflaxseed through the actions of the lignan SDG is protective againstradiation-induced tissue damage in-vivo.

Increased generation of reactive oxygen species (ROS) such as superoxideanion (O₂ ⁻), hydroxyl radical (.OH), and hydrogen peroxide leads totissue damage under various experimental and pathological conditions.Reactive oxygen species result in cellular damage by oxidativemodification of cellular membrane lipids, proteins and the genomic DNA.A number of studies have shown that extracted, purified or syntheticflaxseed SDG is a potent anti-oxidant in vitro as well as in vivo.Therefore, SDG as an antioxidant has therapeutic potential under variousexperimental and disease conditions including radiation-induced tissuedamage in patients undergoing radiation therapy.

Polyphenols commonly occur as glycosides in plants and possessantioxidant properties. Flavonoids, as antioxidants, interfere with theactivities of enzymes involved in generation of reactive oxygen species,quenching of free radicals, chelating transition metals and renderingthem redox inactive in the Fenton reaction. Secoisolariciresinol (SDG)is the major lignan in flaxseed and has been shown to be a potentanti-oxidant in vitro as well as in vivo. In order to explore thetherapeutic potential of flaxseed lignan secoisolariciresinol (SDG), SDGwas synthesized by a chemical reaction using vanillin as a precursormolecule and antioxidant properties of the synthetic SDG (R,R) and SDG(S, S) was determined by assessing their reducing power, metal chelatingpotential, and free radical scavenging activity for hydroxyl, peroxyland DPPH radicals. In the present example, we have investigated theradioprotective characteristics of synthetic SDG (R,R), SDG (S, S)enantiomers and a commercially available SDG (as control) by assessingtheir potential for preventing γ-irradiation-induced damage to plasmidDNA (pBR322) and calf thymus DNA. Radiation-induced damage to plasmidDNA was assessed by the increase in open coiled form of plasmid DNA anddecrease in super coiled form of the plasmid DNA. Radiation-induceddamage to genomic DNA was assessed by determining the level of DNAfragmentation. In this example, we have examined the efficacy ofsynthetic SDG (R,R), SDG (S,S) and commercial SDG againstradiation-induced DNA damage in a cell-free system.

The antioxidant properties of the SDG molecule have been previouslydemonstrated. We have shown that natural, commercially available SDG haspotent free-radical scavenging properties in cells exposed to gammaradiation. The antioxidant and free radical scavenging characteristicsof these synthetic SDG (R,R) and SDG (S,S) enantiomers were investigatedand have been demonstrated to possess strong reducing power, highmetal-ion chelating potential, and high free radical scavenging activityfor hydroxyl, peroxyl and DPPH radicals. These characteristics of thesynthetic SDG (R,R) and SDG (S,S) indicate that these molecules showstrong potential for modulating cellular redox state, decreasingmetal-ion concentration, and scavenging oxygen free radicals. Thesecharacteristics of the synthetic SDG enantiomers suggest their abilityto function by acting at and preventing all the three steps ofinitiation, propagation as well as termination of the free radicalreaction, suggesting that these underlying mechanisms are potentiallyresponsible for the radioprotective characteristics of the SDG (S,S) andSDG (R,R) enantiomers in vivo.

One observation that was made is that the maximum radioprotection ofgenomic DNA by SDG is already achieved at approximately 5.0 μMconcentration which is well below the EC₅₀ values for their free radicalscavenging and antioxidant effects. Therefore, SDG as an antioxidant andfree radical scavenger can also function as a DNA radioprotector andradiation mitigator.

In summary, in the present example, synthetic SDG (S,S) and SDG (R,R)enantiomers were demonstrated to possess strong radioprotectioncharacteristics. The radioprotection potential of these enantiomers wasdetermined using plasmid DNA (pBR322) and calf thymus DNA. The syntheticSDG (S,S) and SDG (R,R) enantiomers prevented the radiation-induceddamage to plasmid DNA in a concentration-dependent manner. Syntheticenantiomers SDG (S,S) and SDG (R,R) also prevented the radiation-inducedfragmentation of calf thymus genomic DNA. At the concentration of 5 μM,SDG (S,S) and SDG (R,R) completely prevented the radiation-inducedgeneration of low mol. wt. fragments of calf thymus DNA demonstratingstrong radioprotective characteristics possessed by these enantiomers.

Example 2 Radioprotective Properties of the Lignan SecoisolariciresinolDiglucoside (SDG) in Lung Cells

Radiation injury to cells is initiated by the generation of reactiveoxygen species (ROS). The spectrum of the damage inflicted by ROS to thecellular machinery includes lipid peroxidation, DNA-protein crosslinks,base modifications, adduct formation and single- and double-strandbreaks (DNA SSBs and DSBs). These modifications have been implicated inradiation-induced apoptosis and cell death. Since cellular DNA damage isa known determining factor in radiation-induced cell death, significantefforts have been made to identify and exploit agents which can protectDNA against radiation damage through interfering with free radicalreactions or by modulating radiation-induced apoptosis.

Prevention from radiation-induced genotoxicity can be achieved by thepresence of antioxidants in the system at the time of exposure. Sinceantioxidants can be ROS scavengers that interfere with free radicalchain reactions, it is possible to protect cellular DNA fromradiation-induced oxidative stress by supplementation with antioxidants.A number of synthetic and natural antioxidant compounds have beenstudied for their radioprotective efficacy. However most of them exhibitinherent toxicity and side effects at their effective concentrations, orhave short shelf life and low bioavailability. Hence the search foreffective and non-toxic radioprotectors has led to investigations intodietary antioxidants and nutraceuticals.

We have evaluated the protective effects of dietary flaxseed (FS)supplementation in preclinical murine models of oxidative lung damagesuch as hyperoxia, acid aspiration injury, and ischemia/reperfusioninjury. We determined that the protective effects of FS may be due inpart to its ability to enhance antioxidant enzyme expression in lungtissues. Importantly, dietary FS ameliorated the adverse effects ofthoracic radiation when given both prior to exposure as well aspost-exposure. In these studies, dietary flaxseed decreasedradiation-induced oxidative lung tissue damage, decreased lunginflammation and prevented pulmonary fibrosis.

Earlier reports suggested that the diversified action of flaxseed mightbe attributed to its lignans which have been shown to possessantioxidant, anti-inflammatory and anti-carcinogenic effects.Secoisolariciresinol diglucoside (SDG) is the prominent FS lignan (about1% of dry weight), which possibly contributes to the beneficial healtheffects of FS grain. SDG is metabolized in the intestine to mammalianlignans, i.e. enterodiol (ED) and enterolactone (EL) by intestinalbacteria. SDG was shown to be beneficial in the treatment of number ofpre-clinical models of diseases such as atherosclerosis and diabetes.SDG has also been reported to exert cardioprotective effects in animalmodels. FS lignans are protective against diverse cancer types assummarized in a recent review on the health effects of SDG by Adolphe etal (Br J Nutr 2010, 103:929) and reported to reduce melanoma metastasisin animals.

In addition, the antioxidant and free radical scavenging properties ofSDG are well documented, which is of paramount importance as the freeradical scavenging ability of a compound can be directly related to itsradioprotective efficacy. In our studies on lung endothelial cells, SDGexhibited free radical scavenging properties when cells were exposed togamma-irradiation while the entire flaxseed lignan component (FLC)enriched in SDG, mediated radioprotection and radiation mitigation inmice. However, characterization of the radioprotective properties of SDGhas not been established.

This study was performed to determine the radioprotective ability of FSlignan SDG and to explore the possible mechanisms responsible for itsaction. The first aim of this study was to evaluate role of SDG onradiation-induced clonogenic death in primary murine lung cells,specifically in epithelial, endothelial cells and fibroblasts. Sinceradiation-induced reproductive death of cells is directly related tocellular DNA damage, we assessed whether SDG can protect cells fromradiation-induced DNA strand breaks by using alkaline comet assay (SSBs)and formation of γ-H2AX foci (DSBs). Furthermore, we examined the effectof SDG pre-treatment in preventing murine primary lung cells fromIR-induced cell death. A number of studies have demonstrated the role ofthe pro-apoptotic protein Bax (Bcl-2-associated X protein) in IR-inducedcell death. We also analyzed direct effect of SDG on Bax and itsantagonist Bcl-2 (B cell leukemia/lymphoma 2) mRNA expression todetermine whether the mechanism of SDG protection involves a shift inthe ratio of these key regulators of apoptosis. Our findings identifythe lignan SDG, a potent bioactive ingredient in FS, mediatesradioprotection in lung cells thus providing novel insight into theradioprotective effects of FS.

Material and Methods Reagents

Secoisolariciresinol Diglucoside (SDG) is commercially available(ChromaDex, Inc., CA). Comet assay kit was purchased from Trevigen,Inc., (Gaithersburg, Md.). P-Histone H2AX (rabbit mAb) was purchasedfrom Cell Signaling Technology, Inc., (Danvers, Mass.). Phosphatebuffered saline (PBS), Bovine serum albumin (BSA), Dulbecco's modifiedEagle's medium (DMEM) with L-glutamine, glucose 1 g/l, without sodiumbicarbonate), HEPES buffer, trypsin, bovine serum albumin (BSA),ethylenediamine tetra acetic acid (EDTA), 4,6diamidino 2-phenyl indole(DAPI), Fetal bovine serum (FBS), Collagenase, Triton-X 100 and Dispasewere purchased from Sigma-Aldrich, St. Louis, Mo., USA.

Cell Lines

Fibroblasts and endothelial cells were isolated from C57/b16 mouse. Forfibroblast isolation, mouse lungs were harvested, minced, and incubatedwith dispase (2 mg/ml) for 45 minutes. Pieces were plated out andfibroblasts were cultured as described previously and used betweenpassages 3 and 10. Pulmonary microvascular endothelial cells (PMVEC)were isolated from murine lungs as described previously. Briefly,freshly harvested mouse lungs were treated with collagenase followed byisolation of cells by adherence to magnetic beads coated with mAb toplatelet endothelial cell adhesion molecule (PECAM). Epithelial cells(C10) cells were originally derived from a normal BALB/c mouse lungexplant and are non-tumorigenic, contact-inhibited, and have alveolartype 2 cell features at early passage.

Clonogenic Survival

Exponentially growing cells were plated as single cells and incubatedovernight. Cells were treated with various doses of the lignan SDG(10-50 μM) 6 h prior to irradiation (2, 4, 6 and 8 Gy). Lignan dose wasselected based on animal studies to be within the physiological levelsreached in the blood circulation when 10% Flaxseed is ingested. Cellswere irradiated with a Mark 1 cesium (Cs-137) irradiator (J. L.Shepherd, San Fernando, Calif.) at a dose rate of 1.7 Gy/min Colonieswere stained and counted 10 to 15 days after irradiation and survivingfraction was calculated.

COMET Analysis

Exponentially growing cells were cultured and treated with SDG (50 μM)at different time intervals prior to irradiation (2 Gy). Cells wereprocessed for comet assay as per manufacturer's instructions (Trevigen,Gaithersburg, Md.). Briefly, cells (1×10⁵ cells/ml in PBS) were mixedwith LMAgarose® (1:10, v/v) and immediately pipetted onto CometSlide™.Cells were then lysed (4° C., 30 min) and kept in dark for unwinding(RT). Electrophoresis was done in a horizontal electrophoresis unit at18 volts (200 Amp) for 25 minutes. Slides were washed twice with DW,fixed in 70% ethanol and dried at 45° C. DNA was stained by SYBR green(Trevigen). At least 150 cells were scored per group. Visual analysis ofcells and comet tail length was measured using Comet Image Analysissoftware (Comet Assay W, Perceptive Instruments Ltd, Haverhill, UK).Images were captured on an Olympus IX51 fluorescence microscope using amonochrome CCD FireWire camera.

Immunostaining and Flow Cytometry for γ-H2AX.

For immunostaining of γ-H2AX, cells were plated on glass coverslips(5,000 cells/coverslip), pre-treated (6 h) with 50 iaM SDG andirradiated (2 Gy). At desired time interval, cells were fixed (4%para-formaldehyde), washed and blocked with PBST (PBS+0.1% TritonX-100containing 5% goat serum, 1% BSA). Cells were incubated with γ-H2AXprimary antibody (1:200) overnight at 4° C. followed by washing withPBST (3×5 min) and incubation with secondary antibody (Alexa Fluor® 488,Invitrogen, CA, USA) for 1 hr at RT. Nuclei were counterstained withDAPI and visualized under fluorescence microscope.

For FACS analysis, cells were trypsinized and washed with PBS. Cellswere then fixed (Fix/Perm buffer, eBioscience), for 45 minutes andwashed thereafter using permeablization wash buffer (BioLegend, USA).Cells were resuspended in 200 μl rabbit monoclonal phospho-histoneγ-H2AX (Ser 139) antibody conjugated to Alexa Fluor® 488 (1:100 v/v,Cell Signaling Technology, US) and incubated for 30 min at 4° C. Cellswere washed again with wash buffer and analyzed. The CyAn ADP (AdvancedDigital Processing) flow cytometer (Dako, Denmark) Coulter, Fullerton,Calif.) was used to measure γ-H2AX and positive cells were quantifiedusing Summit Software (Dako, Denmark).

Morphological Detection of Apoptotic Cells

Apoptotic cells are morphologically characterized by condensation ofnucleus and cytoplasm, membrane blebbing, cell shrinkage, and breakdownof nuclear DNA, first in large segments and subsequently in nucleosomalfragments and finally formation of well-enclosed apoptotic bodies.Percentage of cells undergoing apoptosis was determined microscopicallyfrom the slides used for micronuclei detection (cytogenetic damage). Atleast, 500 cells were counted for each experiment (experiment donetwice) and percent apoptotic cells were determined as follows:

% Cell Death=N _(a) /N _(t)×100,

where N_(a) is the number of cells with apoptotic bodies and N_(r) isthe total number of cells analyzed.Gene Expression Analysis by Quantitative Real Time PCR (qPCR)

qPCR was performed using TaqMan® Probe-Based Gene Expression Assayssupplied by Applied Biosystems, Life Technologies (Carlsbad, Calif.). Toevaluate the effect of SDG treatment on the mRNA expression of apoptoticgenes, individual TaqMan® gene expression assays were performed for Bax(Mm00432051_m1) and Bcl-2 (Mm00477631_m1).

Briefly, cells were pre-treated with SDG (50 μM, 6 hrs) and irradiated(2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen,Valencia, Calif.) and quantified using a NanoDrop 2000 (ThermoFisherScientific, Waltham, Mass.). Reverse transcription of RNA to cDNA wasthen performed on a Veriti® Thermal Cycler using the High Capacity RNAto cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad,Calif.). qPCR was performed using 25 ng of cDNA per reaction well on aStepOnePlus™ Real-Time PCR System (Applied Biosystems, LifeTechnologies, Carlsbad, Calif.). Gene expression data was normalized to18S ribosomal RNA and calibrated to untreated control samples accordingto the ΔΔC_(T) method.

Apoptosis Detection by Western Blotting

Apoptosis was determined in mouse lung epithelial cells by levels of Bax(an apoptosis promoter), Bcl-2 (an apoptosis inhibitor), cleavedcaspase-3, and cleaved poly (adenosine diphosphate-ribose) polymerase(PARP) seen using immunoblotting. Briefly, cells were lysed in PBScontaining protease inhibitors Immunoblot analysis of cell lysates wasthen performed using 10 well SDS 12% NuPAGE gel (Invitrogen, CarlsbadCalif.). Electrophoresis was performed at 200V for 1 hour. Transfer toPolyScreen PV transfer membrane (PerkinElmer Life Sciences, Boston,Mass.) was performed for 1 hour at 25 volts. Membrane was blockedovernight in 5% non-fat dry milk in phosphate buffered saline. Thenon-fat dry milk was then discarded and the membrane was incubated withprimary antibody. Protein levels of Bax, Bcl-2, cleaved caspase-3, andcleaved PARP were detected using manufacturer recommended dilutions(Cell Signaling Technology, Danvers, Mass.) using rabbit anti-mousemonoclonal antibody against BAX and Bcl-2, and rabbit anti-mouse cleavedcaspase-3 (Asp175), monoclonal antibody and a rabbit polyclonalanti-cleaved PARP (214/215) cleavage site specific antibody. Themembrane was washed five times and then incubated in secondary antibodyconjugated to horseradish peroxidase for 45 minutes at room temperature.Membranes were developed using Western Lighting ChemiluminescenceReagent Plus (PerkinElmer Life Sciences, Boston, Mass.) and quantifiedby densitometry scanning of specific bands (20 kDa for Bax, 26 kDa forBcl-2, 17/19 kDa for cleaved caspase-3, and 89 kDa for cleaved PARP)that were adjusted for loading using β-actin expression level detectedby specific secondary antibody (Sigma, St. Louis, Mo.).

Statistics

Results are expressed as mean±SEM. Survival curve for clonogenic assaywas prepared using KaleidaGraph software (4.0). Statistical differencesamong groups were determined using one-way analysis of variance (ANOVA).When statistically significant differences were found (p<0.05)individual comparisons were made using the Bonferoni/Dunn test (Statview4.0).

Results SDG Treatment Increases Colony Forming Ability of IrradiatedPrimary Lung Cells

The clonogenic survival assay has been used widely to determine cellularreproductive death after a cell undergoes any genotoxic stress afterexposure to environmental and pharmaceutical carcinogens, ionizingradiation etc. In this example, the effect of SDG (10-50 μM)pre-treatment on radiation-induced reduction in clonogenicity of primarylung cells (epithelial cells, endothelial cells and fibroblasts,respectively) was evaluated. Results show that SDG (10-50 μM) alone didnot elicit any adverse effect on the colony forming ability of all thethree cell types as compared to their respective untreated control cells(100%) (FIG. 1A-1C).

Radiation treatment significantly (p≦0.01) reduced the colony formingability of epithelial and endothelial cells in a dose-dependent manner.When cells were treated with SDG prior to irradiation, the survivingfraction was enhanced significantly in all the treatment groups (FIG.7A, 7B). Maximum protection against radiation-induced loss inclonogenicity in fibroblasts was observed in 50 μM SDG pre-treatedirradiated group (FIG. 7C). Therefore, we selected this particularconcentration of SDG for our further studies.

SDG Prevents Formation of DNA SSBs in Irradiated Primary Lung Cells

We first performed a study to determine the kinetics of DNA damage inall cell types (endothelial, epithelial, fibroblasts) following aradiobiologically relevant dose of 2 Gy. As expected, radiation exposureinduced significant DNA damage, as evidenced by the increased tailmoment, in all cell types compared to their respective non-irradiatedcontrol cells. The extent of DNA damage was maximum at 30 minutespost-irradiation. The extent of DNA damage was decreased as timepost-irradiation reached up to 60 minutes and further steeply declinedby 2 h post irradiation. Hence, we opted for 30 minutes time intervalfor further studies related to radiation induced DNA damage (FIG. 8A).

Radiation exposure (2 Gy) led to a significant increase in comet taillength in comparison to their non-irradiated control cells. Cells weretreated at various time intervals (0 h, 2 h, 4 h, 6 h and 24 h) prior toirradiation. Pre-treatment of cells with SDG (50 μM), at all timeintervals, significantly inhibited radiation-induced comet tail lengthin all three types of lung cells. However, maximum protection againstradiation-induced tail moment of DNA in all cell types was observed at 6h SDG treatment prior to irradiation (FIG. 8B). A representativefluorescence photomicrograph depicting the formation of comet tails, inirradiated (in presence and/or absence of SDG) lung epithelial cells,has been shown in the Insert, FIG. 8B.

SDG Treatment Abrogates the Formation of γ-H2AX Foci in Murine PrimaryLung Cells

To further test our hypothesis whether SDG can protect against oxidativeDNA damage, we also evaluated the action of SDG on induction of γ-H2AXfoci. The effect of SDG pre-treatment on oxidative DNA damage, evidencedby γ-H2AX formation in murine primary lung cells after irradiation wasevaluated using both standard microscopy-generated image analysis (FIGS.9, 10) and flow cytometry (FIG. 11) methodologies. Results offluorescence microscopic analysis show that radiation (2 Gy) exposureled to a significant increase in the formation of γ-H2AX foci in allthree cell types (FIG. 9). The number of foci/cell increasedsubstantially by 15 minutes, peaked at 30 min post irradiation(46.7%±0.5, 33.6%±3.2 and 30.0%±1.4 of γ-H2AX-positive cells, forepithelial, endothelial and fibroblasts, respectively) while numbersdecreased notably within 1 h of exposure albeit still significantlyhigher than non-irradiated control cells. All values were significantlyhigher (FIG. 9) compared to their respective non-irradiated controlcells (p<0.005 for all cell types).

SDG pre-treatment (6 hours prior IR) significantly decreased theinduction of γ-H2AX, as the number of γ-H2AX positive cells decreased to22.7%±2.17, 21.92%±2.88 and 22.1%±1.9 in irradiated epithelial cells,endothelial cells and fibroblasts, respectively (p<0.005 for epithelial,and p<0.05 for endothelial and fibroblasts). The ability of SDG toprotect cells from the formation of γ-H2AX foci appeared to beindependent of cell type as pre-treatment of SDG protected all threetypes of lung cells from radiation-induced DNA strand breaks. FIG. 10depicts a representative fluorescence photomicrograph of microscopicanalysis of γ-H2AX positive cells in primary lung epithelial cells.

The protective effect of SDG on blunting the induction of γ-H2AXpositive cells after radiation exposure was further confirmed using flowcytometry. As expected, a similar pattern in the induction of γ-H2AXpositive cells was observed post-irradiation; however, number of γ-H2AXpositive cells was significantly abrogated by SDG pre-treatment in allcell types (FIG. 11).

SDG Treatment Prevents Primary Lung Cells from IR-Induced ApoptoticDeath

To study the cytoprotective effect of SDG in terms of apoptosis, nucleiwere stained with DAPI for visualization by microscopy and counted. Themicroscopic analysis demonstrated that the control cells had intactchromatin (apoptotic cells ˜4-5%), whereas radiation exposuresignificantly (p≦0.05) increased the percentage of apoptotic cells in atime and dose dependent manner. At 24 h, percentage of apoptotic cellswas observed as 13.9%±1.08 and 15.1%±1.95 in epithelial and endothelialcells, respectively. SDG pre-treatment (50 μM) significantly (p≦0.05)countered the IR-induced increment in the percentage of apoptotic cells(9.9%±1.08 and 10.71%±1.45 apoptotic cells in epithelial and endothelialcells, respectively) as evident in FIGS. 12 (A and B). Fibroblasts werefound to be most sensitive as radiation exposure led to notable 36.4%and 41.8% enhancement in apoptotic cells at 24 and 48 hr, respectively.Importantly, as shown with epithelial and endothelial cells,pre-treatment with SDG significantly reduced the extent of apoptosis inlung fibroblasts also (FIG. 12C).

SDG Treatment Modifies the Expression of Regulators of Apoptosis inMurine Primary Lung Epithelial Cells

In order to further elucidate the radioprotective effects of SDG inblunting DNA damage and cell death, we hypothesized that SDG may shiftthe ratio of pro-apoptotic and anti-apoptotic regulator proteins. Wetherefore tested whether SDG treatment of lung cells in the presence orabsence of radiation would modify the gene expression of Bax and Bcl-2and whether these changes would translate to changes in protein levels.For this, lung epithelial, endothelial and fibroblast cells were treatedwith SDG (50 μM) and enzyme mRNA levels evaluated at 6, 24, and 48 hourspost-IR (FIGS. 13A and 13B) by qPCR. We observed that 2 Gy led to anapproximate 11 fold increase in pro-apoptotic Bax mRNA levels at 24 and48 hours post-IR, which was significantly (p<0.05) inhibited by SDGpre-treatment. Alternatively, anti-apoptotic Bcl-2 mRNA levels were notaltered due to radiation exposure, yet increased 6.6 and 3.5 fold in SDGpre-treated cells at 24 and 48 hours post-IR treatment, respectively.Upon protein validation by western blot analysis of changes in Bax andBcl-2 mRNA levels, we found subsequent increases in both Bax and Bcl-2protein levels that trended towards decreased levels in SDG pre-treatedcells (FIG. 13C depicts representative blots and 13D and 13E show banddensity quantification). To further investigate the underlying mechanismbehind the observed radioprotection by SDG, we evaluated the effects ofSDG in modifying levels of key protein implicated in the apoptoticsignaling cascade: executioner cleaved caspase-3 and cleaved PARP.Overall, levels of cleaved caspase-3 and cleaved PARP were significantlyincreased in irradiated cells at 6, 24, and 48 hours post-IR and trendedtowards decreased levels in SDG pre-treated cells (FIG. 14A depictsrepresentative blots and 14B and 14C show band density quantification).

Discussion

In this example, we demonstrated that FS lignan SDG can protect murineprimary lung cells against radiation-induced oxidative DNA damage andapoptotic death. We observed that SDG pre-treatment not only improvedIR-induced cytotoxicity as measured by clonogenic survival, but alsodecreased the induction of DNA strand breaks (DSBS and SSBS) and celldeath in lung cells. Importantly, expression of genes implicated in theregulation of apoptosis was also altered by SDG treatment. Thesefindings suggest that SDG may be useful as a non-toxic radioprotectiveagent in radiation scenario and in improving the therapeutic indices ofradiotherapy.

IR-induced cell death is a classical marker of cellularradiosensitivity, which is characterized by loss in clonogenic survival.Similarly, we also noticed a radiation dose dependent loss inclonogenicity in all three murine lung cells which was attenuatedsignificantly by SDG pre-treatment (FIG. 7). Cellular DNA is the primarytarget of IR-induced damage in the cell. ROS generated during exposureinduces an array of changes in cellular DNA ranging from mutations, baselesions, cross-linking, SSBs and DSBs. Damage in DNA cannot be replacedand thus must be repaired and if it remains unrepaired, the cell mayresort to induction of apoptosis or necrosis. Therefore, the protectionof target cellular DNA confers the first line of defense againstgenotoxic insults. ROS generation occurs within seconds of exposure andpersists for few minutes post-irradiation. Thus early radiationresponses such as immediate DNA damage occur within minutes afterradiation exposure. Similarly, in time dependent kinetic studies ofirradiated cells, we also observed that extent of DNA strand breaks, asevidenced by comet tail length, reached to their maximum level within 30minutes of exposure and lessen down thereafter (FIG. 8A).

The comet assay is a sensitive technique for the detection of DNAdamage/repair at the cellular level and has been used widely toinvestigate DNA strand breaks. Polyphenols have the ability to protectnormal tissue or cells from damaging effects of radiation by reducingROS mediated oxidative DNA damage. In the present study, by employingstandard alkaline comet assay, we also evaluated the role of SDG againstIR-induced SSBs in murine primary lung cells. Our results are concordantwith other reports that show a similar protective effect fromradiation-induced DNA SSB for other phenolics such as, chrysin andepicatechin.

While SSBs are easily repaired by the cell, DSBs are more difficult forcells to repair and are more likely to result in mutagenesis, hence DSBsrepresent mostly the lethal cellular event. H2AX molecules becomephosphorylated (γ-H2AX) along megabase-long chromatin domains for eachDNA double-strand break introduced by irradiation and γ-H2AX loss orde-phosphorylation correlates time-wise with DNA repair. We report herethat SDG protected cellular DNA from IR-induced DSBs in all three typesof cells tested. Our results are in agreement with some other studieswhich also show that polyphenols like Resveratrol and green tea catechinprotect cells from IR-induced DNA strand breaks. However, in the absenceof radiation, SDG did not exert any toxic effect in either of thesecells. As oxidative DNA damage is considered to be a precursor to manycancers, a reduction in such damage by SDG acting as antioxidant maylead to reduced risk of cancer.

It is well known that IR-induced apoptosis in cells and tissues is duein part to the induction of DNA damage in cells. Though severalmechanisms (O6-methylguanine, base N-alkylations, bulky DNA adducts, DNAcross-links) are involved in DNA damage-dependent apoptosis, DNAdouble-strand breaks play a major role in inducing apoptotic cell death.In this study pre-treatment of cells with SDG protected againstradiation-induced apoptosis in lung cells and inhibited comet taillength and γ-H2AX foci formation. Thus, the protective effects of SDGagainst radiation-induced apoptotic cell death could be attributed toits ability to reduce cellular oxidative stress, restoration of ionichomeostasis and its ability to prevent DNA damage. These findings are inagreement with the anti-apoptotic properties of dietary wholegrainflaxseed in our murine studies of ischemia-reperfusion andradiation-induced tissue damage and point to the SDG as the likelybioactive component in flaxseed mediating the protective, anti-apoptoticeffects in tissues.

Because IR reduces the level of antioxidant enzymes in cellular milieu,the pre-treatment of cells by an antioxidant might further interferewith ROS and thereby decreasing the risk of interaction of ROS withbio-molecules. In the previous example, we have reported earlier thatpre-feeding of animals with a diet containing flaxseed lignan complexincreased the levels of protective phase II antioxidant enzymes in mouselungs. The postulated mechanism for these actions involves theactivation of antioxidant response element (ARE) mediatedtranscriptional induction. Further, Nrf2 also initiates the de novosynthesis of various antioxidant enzymes responsible for protectionagainst oxidative stress mediated cytotoxicity. Some other reports alsosuggest that polyphenols like curcumin and EGCG exert protective effectsagainst oxidative stress which is implicated by their ability to inducephase II antioxidant enzymes.

It is evident from the results that SDG reduces cell death in murineprimary lung cells owing to its antioxidant properties and protectiveeffects on DNA. Our findings corroborate with the results of Hseu et al(Food Chem Toxicol 2012, 50:1245) who reported that ellagic acid, apolyphenol found in pomegranate, protects human keratinocytes againstUVA-induced oxidative stress and apoptosis through the upregulation ofthe HO-1 and Nrf-2 genes. Our study provides novel evidence that theradioprotective action of SDG in combating the IR-induced oxidativedamage and apoptotic cell death may be by direct induction of theantioxidant defense system.

Taken together these results demonstrate that the flaxseed lignan SDG inflaxseed has potent radioprotective properties that likely contribute tothe observed protective effects of the wholegrain in animal studies.

Example 3 Secoisolariciresinol Diglucoside (SDG) Scavenges HypochloriteIons: A Novel Mechanism of SDG Protection of Genomic DNA from Radiation

Hypochlorous acid (HOCl), a potent oxidant, is produced by neutrophilsby activated myeloperoxidase which catalyzes the reaction betweenphysiologically present chloride ions and hydrogen peroxide (H₂O₂).Activated neutrophils produce H₂O₂ and superoxide anion O₂.⁻. Atphysiological pH, a mixture of both HOCl and hypochlorite ion (ClO⁻)exists. HOCl kills microorganisms by oxidative damage. However,excessive production is known to cause damage to tissues. Hypochloritemodifies adenine nucleotides resulting in formation of chloramines thatappears to be a major mechanism of neutrophil-mediated toxicity.

HOCl and its conjugated base ClO⁻ have been shown to oxidize aminoacids, peptides, proteins and lipids and to chlorinate bases in cellularDNA and RNA. The reaction of HOCl/ClO⁻ results in modification of bothpurine and pyrimidine nucleotides at the endocyclic —NH groups ofguanine and thymine as well as the exocyclic NH2 groups of guanine,adenine and cytosine derivatives resulting in the formation ofchloramines such as (RNHCl) and RR′NCl. The primary modified bases werefound to be 5-chlorocytosine, 8-chloroadenine and 8-chloroguanine in DNAand RNA of SKM-1 cells.

It is well known that γ-radiation is capable of ionizing atoms andmolecules. In biological systems or in solution, ionizing radiationgenerates hydroxyl radicals (.OH) which are believed to be the source ofionizing radiation-induced damage to cellular components includinglipids, proteins and DNA. However, these highly unstable radicals can bescavenged by ions which are present in the physiological medium at veryhigh concentrations. This leads to generation of reactivechlorine-containing intermediates, among which relatively stable ClO⁻ isthe radiation-derived toxicant. In chloride-containing solutions, theClO⁻ and other active chlorine derivatives of oxidative nature areformed as products of radiolysis; they can contribute to suppression ofphysiological functions of organisms. Therefore, we propose thatradiation-induced DNA or protein damage is mediated, in part, byradiation-generated ClO⁻.

Chemically synthesized two diastereomers of SDG have recently been shownto be equipotent in their antioxidant, free radical scavenging and DNAprotective properties. The present study evaluates SDG in DNAradioprotection from γ-radiation induced generation of ClO⁻ inphysiological saline solutions using highly novel and specificfluorescent probes. Hypochlorite-specific 3′-(p-aminophenyl) fluorescein(APF) and hydroxyl radical-sensitive 3′-(p-hydroxyphenyl) fluorescein(HPF) provide greater specificity and reproducibility for determinationof the above reactive oxygen species (ROS).

Materials and Methods Chemicals

ROS indicator probes APF and HPF, plasmid DNA (pBR322), and 1 kb plusDNA ladder were from Invitrogen (Life Technologies, Carlsbad, Calif.).

Determination of Hypochlorite

The fluorescence of ROS probes APF and HPF in PBS was measured atexcitation/emission at 490 nm/515 nm in presence of hypochlorite orafter γ-radiation exposure. Data is expressed as relative fluorescenceunits (RFU).

γ-Radiation-Induced Generation of Hypochlorite by Determining TaurineChloramine

Chlorination of taurine was determined using TMB assay. The data isexpressed as taurine chloramine (absorbance) as well as ClO⁻concentration (μM).

Hypochlorite-Induced Damage to Calf Thymus and Plasmid DNA

Calf thymus or plasmid DNA was incubated with hypochlorite for 2 hrs at37° C. DNA samples were subjected to agarose (1%) gel electrophoresisand analyzed.

Determination of Hypochlorite-Induced Chlorination of 2-Aminopurine(2-AP)

2-AP in PBS was exposed to hypochlorite and fluorescence spectrarecorded between 360-390 nm with the emission maximum at 374 nm. The %change in 2-AP calculated.

Statistical Analysis of the Data

The data obtained are presented as mean values±standard deviation. Thedata were subjected to one-way analysis of variance (ANOVA) withpost-hoc comparison using Bonferroni correction using Statview Program.P value≦0.05 was considered as significant.

Results

In this study we investigated the ability of SDG to scavengeradiation-induced ClO⁻, as a potential mechanism of DNA protection fromradiation exposure in physiological solutions.

SDG Scavenges Hypochlorite Ions

The specificity of the selected fluoroprobes was evaluated using sodiumhypochlorite. FIG. 15A shows a linear increase in APF fluorescenceintensity with an increase in ClO⁻ concentration (1-4 μM). Importantly,HPF fluorescence intensity increased only marginally, indicating thatAPF fluorescence is mainly ClO⁻-dependent. ClO⁻ doses were selected tobe within this range for subsequent experiments. The ability of SDG toscavenge ClO⁻ by SDG (commercially available) was then evaluated.Indeed, SDG decreased ClO⁻ dose-dependently (FIG. 15B). Lastly, weevaluated the ClO⁻ scavenging effect of synthetic SDG diastereomersSDG(S, S) and SDG (R, R). At 0.5 μM, SDG (R,R) and SDG (S,S), and SDG(com) scavenged ClO⁻ (FIG. 15C) with a comparable potency that wassimilar to silibinin, an established ClO⁻ scavenger.

SDG Scavenges γ-Radiation-Induced Hypochlorite

Radiation generates ClO⁻ dose-dependently, as evidenced by an increasein fluorescence intensity of APF. Specifically, 50 Gyγ-radiation-induced APF and HPF (FIG. 16A) which was dose-dependentlydecreased by SDG (FIGS. 16A and B). The initial slope for decrease forAPF fluorescence (1,816.30) was higher compared to HPF (695.75)indicating a selective scavenging effect of SDG on ClO⁻ (insert, FIG.16A). The effect of SDG on APF was significantly more pronounced ascompared to HPF (FIG. 16B). SDG blunted ClO⁻ generation from radiationexposure (25 and 50 Gy) shown by increased APF (FIG. 16C) and HPF (FIG.16D) fluorescence (p<0.05). The ratio APF/HPF decreased by SDGindicating selective scavenging of ClO⁻.

Radiation Dose-Dependent Increase in Hypochlorite as Chlorination ofTaurine

To establish that radiation-induced ClO⁻ chlorinates —NH groups inbiological molecules, we evaluated radiation-induced chlorination oftaurine. The results (FIG. 16E) show that in normal saline, γ-radiationsignificantly increased taurine chloramine formation at 50, 100, and 200Gy indicating that γ-radiation induces ClO⁻ generation which wasdependent in chloride concentration (FIG. 16F). The results providestrong evidence that radiation induces ClO⁻ in a physiological solution,capable of damaging biomolecules.

SDG Protects Hypochlorite-Induced Damage to Calf Thymus and Plasmid DNA

We determined whether ClO⁻ induces damage to genomic (FIGS. 17A-D) andplasmid (FIGS. 17E-F) DNA. Indeed ClO⁻ induces dose-dependent DNAfragmentation (FIGS. 17A, B) with an increase in low molecular weightfragments. Damage to genomics DNA exposed to 0.5 mM hypochlorite 1.0 μMwas blunted by all SDGs (commercially available or synthetic), to levelscomparable to quercetin, a known antioxidant and silibinin, a ClO⁻scavenger (FIG. 17C, D). Similarly, damage by ClO⁻ to plasmid DNA wasblunted by SDG (FIG. 17E, F). Specifically, we evaluated amounts ofsuper coiled (SC) plasmid DNA as compared to damaged, open coiled-DNAwhich have a different mobility pattern in agarose gel electrophoresis.The presence of SDG at (25 μM) decreased the ClO⁻-induced damage toplasmid DNA and preserved the DNA in mostly (81.3%±9.4%) super coiledform as compared to OC (18.6%±9.4%). Protective Effect of SDG onHypochlorite-Induced Chlorination of 2-AP

To determine whether the ClO⁻ damage to DNA occurs by nucleobasemodification, we evaluated ClO⁻-induced chlorination of 2-AP, afluorescent analog of purine. Hypochlorite, given at comparableconcentrations (10 μM) to those generated by radiation exposure (FIGS.15A, 16A, 17C) decreased 2-AP fluorescence which was prevented bypre-treatment (60 seconds) with SDG (FIGS. 18A and B). Most importantly(FIGS. 18B and C), post-treatment with SDG resulted in significantrecovery from hypochlorite-induced 2-AP modification if added at +15,+30, +60, +120, +180 or +300, seconds following the exposure to ClO⁻.These results demonstrate the nucleobase protective characteristic ofSDG against hypochlorite-induced modification of purine bases.

Protective Effect of SDG on γ-Radiation-Induced Chlorination of 2-AP

To determine whether radiation induces nucleobase chlorination, we usedthe same system as above, namely 2-AP fluorescence (FIG. 19A). Indeed,exposure to γ-radiation resulted in dose-dependent decrease influorescence intensity (an observation similar to that in presence ofClO⁻, (FIG. 18A) which was prevented by SDG (FIGS. 19A and B). Theseresults demonstrate that γ-radiation induces chlorination of anucleobase and established protective properties of SDG against suchradiation-induced modifications of purine bases.

Proposed Mechanism of SDG Action in Preventing or Mitigating NucleobaseChlorination by Radiation

Regarding the mechanism of recovery or prevention of N-chlorinatednucleobases, we suggest either a two-electron reduction ofN—Cl-molecules by electron-rich aromatic ring or a one-electronreduction, leading to formation of a free N-radical, which, in turn,captures hydrogen atom from —OH group in phenolic SDG moieties. Scheme 1(FIG. 20) indicates the proposed mechanism of SDG protection. Theresults of our present study provide the evidence, for the new mechanismof radioprotective action of SDG by scavenging hypochlorite ions andprotecting from radiation-induced DNA damage.

Discussion

The results of this study provide evidence for the following: i) SDG, aknown lignan antioxidant and free radical scavenger, detoxifiedhypochlorite ions generated in physiological solutions by chemical meansas well as by radiation; ii) SDG protected genomic DNA as well asplasmid DNA from hypochlorite-induced damage; iii) The mechanism of SDGdefense (protection or recovery from hypochlorite-induced DNA damage),involves scavenging hypochlorite and regeneration of amino (—NH) groupson nucleobases from chloramines (—NCl); iv) Exposure to γ-radiationresulted in increased taurine chloramine formation; v) Exposure toγ-radiation resulted in increased chlorination of a purine base that wasprevented by SDG; vi) SDG action is equally effective in protecting DNAfrom hypochlorite-induced damage when added prior to or post-exposure,i.e., it can act as a protector and/or mitigator of nucleobasechlorination; vii) Synthetic SDG (S,S) and SDG (R,R) diastereomers wereequally potent in scavenging hypochlorite ions and preventinghypochlorite-induced DNA damage as compared to commercially availableSDG, silibinin, and quercetin (a natural antioxidant flavonoid). Theseresults demonstrate the protective and mitigating property of SDG forhypochlorite-induced modification of nucleobases.

Using a mouse model of thoracic radiation damage, we have establishedthe tissue radioprotective role of whole grain dietary flaxseed, a grainrich in lignan polyphenols, as well as of flaxseed lignan formulationsenriched in SDG. These studies emphasized the radioprotective andradiation mitigating properties of the lignan SDG againstradiation-induced tissue damage in vivo. Extracted, purified orsynthetic flaxseed SDG is a potent antioxidant in vitro as well as invivo. In order to explore the therapeutic potential of SDG we havesynthesized SDG by a novel chemical reaction and determined theantioxidant properties of the synthetic SDG (R,R) and SDG (S,S) byassessing their reducing power, metal chelating potential, and freeradical scavenging activity for .OH, peroxyl and DPPH radicals. We alsodemonstrated the radioprotective characteristics of synthetic SDG (R,R),SDG (S,S) diastereomers by assessing their potential for preventingγ-irradiation-induced damage to plasmid DNA (pBR322) and calf thymusDNA.

We have also showed that the maximum radioprotection of genomic DNA bySDG is already achieved at approximately 5.0 μM concentration which isbelow the EC50 values for their free radical scavenging and antioxidanteffects, typically in the range of 130-200 μM. It is interesting to notethat the maximum effectiveness of SDG in scavenging radiation-inducedhypochlorite as determined in this study, falls within 0.5 to 5 μM. Thissuggests that the DNA-protective effects of SDG are due in part toscavenging of damaging ClO⁻. The results showing the protective effectof SDG on hypochlorite-induced modification of 2-AP indicate that SDGprotects DNA by preventing hypochlorite-induced damage to thenucleobases.

HOCl is produced by myeloperoxidase of activated neutrophils usinghydrogen peroxide generated by NADPH oxidase and chloride ions assubstrates. HOCl can chlorinate and oxidize nucleobases. Since HOCl canchlorinate nucleobases, this might cause genotoxicity. Chlorinatednucleosides have been identified and linked to inflammation and cancer.

There could be several potential mechanisms by which SDG may protect DNAfrom radiation-induced damage: 1) by scavenging hypochlorite ions thatcause chlorination and oxidation of nucleobases; 2) by scavenging .OHfree radicals that produce hypochlorite by reacting with chloride ions.We observed that the production of .OH was drastically decreased in thepresence of chloride ion in phosphate buffered saline (PBS) as well asin saline alone (preliminary experiments). Considering the highconcentration of chloride ions in physiological medium in the body, thesignificance of production of hypochlorite and hypochlorite-induceddamage to cellular components including DNA would be a predominantmechanism of radiation damage; 3) by associating with DNA base pairs asseveral flavonoids such as lutiolin, kempferol and quercetin; 4) byblocking abstraction of protons or addition of .OH on the purine andpyrimidine bases especially at C5, C6 and C8, and at the deoxyribosesites. These mechanisms have been proposed for protection from freeradical-induced DNA damage; 5) lastly, by reduction of chloroaminesformed, thus, regenerating internal and external amino groups in nucleicacids. Therefore, SDG as a scavenger of hypochlorite ions as well asbeing an antioxidant and free radical scavenger and protector ofnucleobases from hypochlorite-induced chlorination can function as a DNAradioprotector and as a radiation mitigator.

Hypochlorite exists in solution as a mixture of hypochlorite anion(ClO⁻), hypochlorous acid (HClO) and free chlorine (Cl₂) in pH-dependentamounts. At physiological pH, ClO⁻ and HClO are the predominantmolecules. Unlike strong, single-electron oxidants such as .OH,hypochlorite is a two-electron oxidant, less reactive and more selectivethan .OH. Hypochlorite can chlorinate electron-rich aromatic rings andNH-compounds. It oxidizes primary and secondary alcohols as well asbenzyl methylene groups and tertiary methine groups, and phenols. Thefirst step of the above reactions is chlorination followed byhydrolysis/HCl elimination. SDG contains all the above reaction sitesexcept amino groups that make the SDG molecule a potent hypochloritescavenger. In Scheme 1 (FIG. 20) we proposed a novel mechanism of DNAprotection by SDG using either a 1-electron or a 2-electron reduction ofa nucleobase.

In summary, we have demonstrated that SDG scavenges hypochlorite (ClO⁻)ions and prevents radiation-induced hypochlorite and DNA damage. Sincehypochlorite ions are known to modify DNA bases bychlorination/oxidation and then subsequently resulting in DNA damage,our findings show SDG may be useful as a radioprotector of normal tissuedamage associated with radiotherapy for cancer treatment or accidentalexposure to radiation.

Example 4 Flaxseed and its Lignans Protects Against the Effects of theBenzo-Alpha-Pyrene

Flaxseed, SDG and lignan derivatives mitigate lung tumorigenesis bytobacco and other environmental carcinogens by inhibiting the multi-stepcarcinogenesis process (FIG. 21). In this example, experiments areprovided indicating that the lignan SDG has chemopreventive activitythrough modulation of the Nrf2-regulated Phase II detoxificationpathway, and perhaps other mechanisms, in animal models. The protectiveeffects of SDG are mediated by the direct ROS scavenging and/or indirectantioxidant/anti-inflammatory properties, and decrease of carcinogentoxicity and DNA damage.

Exposure of murine epithelial cells to the tobacco and environmentalcarcinogen benzo-alpha-pyrene (BaP) induces damaging reactive oxygenspecies (ROS) as detected by a redox-sensitive fluorescence dye (FIG.23). As early as 2 hours, post exposure to the carcinogen, a robustincrease of fluorescence intensity indicates ROS generation in cells.

To demonstrate that SDG decreases oxidative DNA damage induced bycarcinogens in cells, SDG (10 μM) was added to human epithelial cells(A549) that were exposed to 25 μM of BaP and oxidative damage to DNA wasdetected using mass spectrometry as indicated by the presence of8-oxo-7,8-dihydroguanine (8-oxo-dGuo). SDG decreased DNA damage at 3 and6 hours post carcinogen exposure (FIG. 22). Likewise, to demonstratethat SDG prevents ROS generation from carcinogen exposure, mouseepithelial cells were exposed to 10 or 20 μM BaP and an increasingconcentration of SDG (0, 0.1, 0.5, 1, 5 μM SDG) and ROS was detected 2hours later. SDG scavenged harmful ROS to negligible levels (FIG. 24).

Similarly, SDG prevents genotoxic stress in human epithelial cellsexposed to BaP. Exposure of cells to a potent carcinogen such as BaP,induces genotoxic stress as indicated by increased levels of p53 protein(FIG. 25). This is mitigated dose-dependently by the presence of SDG, at5, 10, 25 and 50 μM concentration. SDG also prevents oxidative DNAdamages in human epithelial cells exposed to BaP. Exposure of cells to apotent carcinogen such as BaP, induces oxidative DNA damage as indicatedby increased levels of gamma-H2AX, a marker for double-stranded DNAbreaks (FIG. 26). This is mitigated dose-dependently by the presence ofSDG, at 5, 10, 25, 50 and 100 μM concentration. Furthermore, SDGprevents DNA adduct formation in human epithelial cells exposed to BaP.Exposure of cells to a potent carcinogen such as BaP, induces theformation of DNA adducts (FIG. 27). DNA adducts are pieces of DNAcovalently linked to a carcinogen and is directly linked to thedevelopment of malignancy. The DNA adduct levels is decreased by thepresence of SDG or its metabolites ED and EL given alone or incombination.

Flaxseed and its lignans provides protection in a mouse model ofchemical carcinogen-induced lung tumors. Mice (A/J strain) are given 4injections intraperitoneally of the tobacco and environmental carcinogenBaP (once weekly) at 1 mg/Kg dose (FIG. 28). Mice are initiated onflaxseed or lignan diet at the time of exposure. Mice are evaluated atvarious times post exposure to determine tumor burden, mouse weight, andoverall health profile.

Flaxseed Decreases Tumor Burden in Mice:

FIG. 29 presents representative clinical images of murine lungs severalmonths post BaP exposure and dietary flaxseed administration. FIG. 30presents representative H&E-strained lung sections of murine lungsseveral months post BaP exposure and dietary flaxseed administration.Nodules indicated by the arrows from mice fed control diet (top panels)or flaxseed (lower panels) appear smaller in the flaxseed-fed mice. Eachpanel represents a different animal.

Histological murine lung sections were evaluated morphologically usingimage analysis software for overall tumor area and nodule size (FIGS.31A and B). There was a significant decrease in the area of the lungoccupied by tumor in the mice fed a flaxseed diet (p<0.03). Similarly,there was a trend for smaller tumor nodule size. Histological murinelung sections were also evaluated morphologically for overall number oftumor nodules per lung (FIG. 32A) and % tumor invading the lung (FIG.32B). There was a trend for less tumor nodules per lung (A) and lesstumor invading with flaxseed supplementation (B).

Finally, flaxseed supplementation was shown to prevent wasting effectsfrom lung cancer induced by BaP. Animal weight was measuredlongitudinally for 200 days post BaP exposure. Mice fed a flaxseed diet,exposed to BaP showed higher weight than those exposed to BaP on controldiet (FIG. 33).

Example 5

Flaxseed and its lignans protect cells and tissues from asbestos-induceddamage Introduction

Malignant mesothelioma (MM) is a devastating, painful and lethal type ofcancer with no realistic chance for therapy and treatment. Developmentof MM has been linked directly to exposure to asbestos fibers. Recentstudies have indicated that the pathogenesis of asbestos-induced cancersis due to chronic inflammation and oxidative tissue damage caused bypersistent asbestos fibers. Whole grain Flaxseed (FS) has knownantioxidant, anti-inflammatory and cancer chemopreventive properties. Inthis Example, the ability of FS and its lignan component (FLC) enrichedin the lignan secoisolariciresinol diglucoside (SDG) given in diets toprevent acute asbestos-induced inflammation and inflammatory cytokinerelease was tested in Nf2^(+/mut); Cdkn2a^(+/mut) mice.

Materials and Methods Mouse Diets and Asbestos Exposure

FS and its lignan component, (FLC) enriched in the lignansecoisolariciresinol diglucoside (SDG) was given in rodent chow. Mice(Nf2^(+/mut);Cdkn2a^(+/mut)) were, placed a day later (+1) or prior (−1)to a single ip bolus of 400 mg of crocidolite asbestos on 10% FS or 10%FLC supplemented diets and evaluated 3 days later for abdominalinflammation and proinflammatory cytokine release (see FIG. 34). The NF2mouse strain was selected as it develops an accelerated form of MM whenexposed to asbestos.

Tissue Harvest and Analysis

Using liquid chromatography, tandem mass spectrometry (LC/MS/MS),systemic levels (i.e., plasma) of flaxseed lignan metabolites such asthe mammalian lignans Enterolactone (EL) and Enterodiol (ED) wereevaluated to ensure that FS was effectively metabolized by the gut floraof this mouse strain and that levels were comparable to those in othermouse models.

Abdominal lavage was performed using PBS and levels of macrophages (MF)and neutrophils (PMN) determined using cytospin analysis.

Discussion

Using liquid chromatography, tandem mass spectrometry (LC/MS/MS),systemic levels (i.e., plasma) of flaxseed lignan metabolites such asthe mammalian lignans Enterolactone (EL) and Enterodiol (ED) wereevaluated to ensure that FS was effectively metabolized by the gut floraof this mouse strain and that levels were comparable to those in othermouse models (FIG. 37). Abdominal lavage levels of macrophages (MF) andneutrophils (PMN) indicated that both FS and FLC blunted acute abdominalinflammation induced by asbestos (FIGS. 36 and 38). In addition, thelevels of pro-inflammatory cytokines TNF-α and IL-1βwere also decreasedby the dietary agents (FIGS. 37 and 38).

Conclusions: These findings indicate that the chemopreventive propertiesof Flaxseed and its lignan component extend to protection fromasbestos-induced tissue and cellular damage. Flaxseed can thus, be usedas a dietary agent in the chemoprevention of malignant mesothelioma.

Example 6 Optimizing Dose and Kinetics of SDG Administration In Vivo

In this Example, data is presented on pharmacokinetics, bioavailability,and dose response in mice given SDG via oral gavage in a water-solubleform.

Dosing Study:

SDG doses of 5, 25, 50 and 100 mg/Kg body weight were administeredorally to mice (n=5 per dose) and 4 hours later tissues were collectedfor analysis. Plasma concentrations of SDG and its bioactive metabolitesED, EL and SECO were analyzed by liquid chromatography and massspectrometry and are expressed in ng/mL (FIG. 39) while gene expressionlevels of Nrf2-regulated antioxidant enzymes (AOE), an intracellulartarget of SDG and its metabolites, were determined in lung tissues fromthe same animals using qRT-PCR (FIG. 40). Gene expression levels of HO1,NQO1 and GST, increased 2-3 fold with as little as 5 mg SDG/Kg andreached an average of 6-fold increase over baseline with 100 mg/Kg SDG.Gene levels are supported by lower but yet significant increase inprotein levels in lung tissues.

Pharmacokinetic Study:

After selecting the 100 mg/kg dose as a dose that induces significantAOE expression while allowing for SDG to be detected in circulation, apharmacokinetic study was performed to determine bioavailability andbiological effects in target tissues (i.e., lung). Blood samples as wellas tissues (lung, brain, liver, kidneys, spleen) were collected at 15min, 30 min, and 1, 2, 4, 6, 8, 12 hours. SDG levels were determined asabove in plasma and lung (FIG. 41). A single dose of 100 mg SDG/Kg waswell absorbed and intact SDG was detectable in the circulation up to 6hours post administration (FIG. 41A) and in flushed, blood-free lungtissues for 4 hours (FIG. 41B). Plasma and lung tissue concentrations ofSDG reached levels of 0.8 and 12.6 μM at 30 minutes post-administration,respectively. Remarkably, robust induction of representative AOE geneexpression levels (H0-1, NQO1, GST) were significantly elevated (p≦0.05)over baseline (FIG. 41C). The observed gene expression increasecorrelated with an increase in protein levels determined by westernblotting (not shown). Importantly, SDG at the same dose given twicedaily for 7 days showed neither intolerance nor toxicity. These dataindicate that significant bioavailability and efficiency can be obtainedby this soluble SDG form. The use of SDG in human therapy is thusgreatly facilitated and the toxicity risk is unlikely. Therefore a doseof 50-100 mg/Kg (1 or 2 mg SDG per day) is sufficient to induce theanticipated protective effects in target tissues and can be used forfurther study.

Example 7 Effects of SDG in BaP-Induced Lung Tumorigenesis inNrf2-Deficient Mice on the Initiation or Promotion Phases ofCarcinogenesis

Data from lung injury models show that FS and FS-derived SDG in dietscan upregulate Nrf2 and Nrf2-actived genes and proteins in lungs. Inthis Example, using Nrf2 knockout mice, the hypothesis that activationof this pathway is an important mechanism of the chemopreventive effectsof SDG is tested and it is determined if SDG has activity in both theinitiation and promotion phases of carcinogenesis.

Effects of SDG in Nrf2-deficient mice.

In these experiments, the effects of synthetic SDG given orally in theBaP-induced lung cancer model in wild-type A/J mice are compared toNrf2-deficient mice backcrossed on an A/J background (colony currentlymaintained at our animal facility). Briefly, one group of wild-type (WT)mice and one group of knockout (KO) mice are fed a control diet. Onegroup of WT mice and one group of KO mice are given SDG via theirdrinking water to achieve 50 and 100 mg/Kg daily SDG consumption (totest dose response relationship in inhibiting the development of lungcancer) as shown in FIG. 42A. SDG dosing studies and kinetics determinedthat SDG should be given for at least a few days, as this was determinedto be the minimum time required for the diets to reach steady state intissues, followed by 4 weekly injections of 1 mg/mouse Benzo[a]pyrene(BaP) given i.p. Both 50 and 100 mg/Kg SDG given orally (oral gavage orin drinking water) can induce Phase enzyme expression in lungs (see FIG.41). Mice are sacrificed at 4, 6 and 9 months (see FIG. 42B) and lungtissues are harvested for a) histological evaluation of tumor burden andquantification by image analysis, b) western blot detection ofNrf2-regulated AOE expression and oxidative stress, c)8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels in murine tissuesand urine, and d) DNA adduct formation. A subset of mice are evaluatedfor lung inflammation using a) Bronchoalveolar lavage, and b) FACSanalysis of inflammatory cells such as neutrophils, activatedmacrophages and T cells using antibodies to CD11b/Ly6G, CD11b/F4/80, andanti-CD3, respectively.

Statistics: The minimum number of mice required per group to achievestatistical significance and suggested 20 mice/group.

Analysis: As above (FIGS. 29 and 31 a reduction in the number and sizeof tumors is expected in the WT mice getting both 50 and 100 mg/Kg SDG,however results are more profound with the 100 mg/Kg dose (less tumor).A higher number of tumors is expected in the Nrf2 KO mice gettingcontrol (no drug) due to loss of ability to detoxify the BaP whencompared to WT mice on control diet. However, no reduction in tumors isexpected in the Nfr2 KO mice receiving the SDG (compared to the KO micegetting no drug), since the primary effect of SDG is mediated via Nrf2activation. Whatever reductions are seen in the tumor burden in theNrf2/SDG mice, is attributable to other mechanisms that are alsocontributing, such as the direct free radical scavenging effects of SDG,which should be detectable by measuring markers of oxidative stress.

Role of SDG and Nrf2 Activation in the Initiation and/or PromotionPhases of Carcinogenesis.

The data above is expected to provide clear mechanistic data regardingSDG administration during both the initiation and promotion phases ofBaP. It is also of interest to determine the efficacy of SDGsupplementation during just the initiation phase (FIG. 42A, Plan B) orduring just the promotion phases (FIG. 42A, Plan C). Accordingly, theexperiments outlined in this schema are performed in wild-type A/J miceusing these different feeding schedules.

Analysis: Decreases in tumor numbers and size are expected when givingSDG during either promotion or initiation. The studies are repeated inNrf2 KO mice, as above, to define the contribution of Phase II enzymeupregulation to this activity. It has recently been reported that Nrf2has two roles during carcinogenesis, one of which is preventive duringtumor initiation and the second that promotes malignant progression.These findings suggest use of Nrf2 to prevent malignant progression inlung cancer, whereas Nrf2 activators are more suited for lung cancerprevention. SDG is expected to be effective as a chemopreventive agent.Notwithstanding the role of Nrf2 in the anti-carcinogenic effects ofSDG, it is possible that other mechanisms are also involved or even moreimportant such as the modulation of miRNA in systemic fluids (blood) orlung tissues.

The Effects of Administration of an SDG Supplement to Smokers onSystemic and Lung-Specific Biomarkers of Oxidant Stress.

A clinical study of oral administration of SDG with genetic andbiochemical endpoints being genetic and biochemical is designed.

Population Selected: The first group will consist of normal, healthyvolunteers who have never smoked. This group allows assessment of truebaseline levels (likely low) of genes of interest and oxidativebiomarkers, in order to evaluate the changes induced by SDGsupplementation in a “non-stimulated” environment. The second group ofsubjects will consist of current, active smokers. This group is selectedbecause: i) they are at high risk for active cancer initiation and arepotential subjects in a chemoprevention trial and ii) it has previouslybeen shown that smoking induces its own genomic changes in therespiratory epithelium, as well as active oxidative stress. This groupallows profiling of genomic and oxidative stress markers in smokers andto determine what genomic changes are induced by SDG in an already“activated” environment. This group also allows a determination of thereduction of elevated markers of oxidative stress by SDG in this highrisk population. A crossover design is selected where each patient willalso ingest both a placebo control and an SDG supplement both given ingelatin capsules (See FIG. 43).

Dosage: A commercial preparation (BREVAIL™) was chosen to avoid themarked variability in SDG content and bioavailability observed withdifferent batches of raw or ground flaxseed. Alternatively, syntheticSDG such as that described by Mishra et al., Bioorganic & MedicinalChemistry Letters 2013, (19):5325 will be used. Pharmacologic studieshad shown that daily dosing with this formulation, which contains 50 mgof SDG, produced ENL levels (median, 63 nmol/L) similar to those foundin the highest quintiles associated with reduction in cancer incidencein case-control studies.

Clinical Study: The study will be a single center, randomized,double-blind, two period cross-over trial. There will be two 3 weektreatment periods interspersed with a one-month washout period (FIG.43). The rationale for the cross-over design is that comparison oftreatments within subjects removes any “subject effect” from thecomparison and enhances the efficiency and power of the study. Twenty(20) healthy volunteers (10 smokers and 10 lifelong nonsmokers) arerandomized to ingest, in order, EITHER 50 mg SDG (either one capsuleBREVAIL™ or synthetic SDG) daily for 3 weeks followed by a 1-monthwashout period and then placebo (same capsule without SDG, provided bythe same provider (Lignan Research, San Diego, Calif.) for an additional3 weeks, OR placebo for 3 weeks, a one month washout, and then 50 mg SDGfor 3 weeks. Oral epithelium (obtained by a mouth swab), exhaled breathcondensate, urine and blood are obtained at baseline, and weeks 2, 3, 7,9, and 10. Active smoking status will be confirmed by urinary cotinineassays. The study will be approved by the Penn Institutional ReviewBoard, and all participants will provide written informed consent. Adetailed questionnaire will be administered at baseline including acomprehensive smoking history, and environmental tobacco exposure (ETS).Participants will be seen weekly for evaluation of side-effects,adherence to test agent ingestion, collection of data on interim tobaccoand medication use and sample collection.

Statistical Rationale/Power Analysis: The major comparisons of interestare the change in urinary oxidative stress, as measured by urinary8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo), changes in exhaledbreath condensate isoprostanes (exploratory), and changes in geneexpression levels in buccal epithelium in study subjects following SDGtreatment. 20 patients is expected to provide reasonable power for thisexploratory study. Because of the nature of this study, corrections arenot applied for multiple comparisons. The study is designed to get anassessment of the effect of the SDG supplement. Note also, that there islimited power to test for carryover effects, but the 4-week washoutperiod should obviate these concerns.

Evaluation of gene expression changes in buccal swab epithelial cellspost oral SDG ingestion

In these experiments, buccal mucosal epithelial cells are used assurrogate tissue to bronchial epithelial tissue. Such cells are easierto obtain (i.e. by using a simple swabbing of the cheek) than bronchialepithelial cells which require an invasive bronchoscopy. The validity ofthis approach in humans is supported by evidence that buccal epitheliumcan serve as surrogate tissue for airway bronchial tissues in gene arraystudies.

This approach was further validated using ingested FS in mice. Aftermice were fed a control or 10% FS diet for three weeks, a range oftissues was harvested and analyzed for expression of two representativeNrf-2 inducible proteins (H0-1 and NQ01) by immunoblot. Marked increasesin these markers in the lung and liver were seen (data not shown).Importantly, clearly detectable increases in nasal epithelium of bothproteins were seen (FIG. 44), thus validating that nasopharyngeal tissuecan also be used as a surrogate for lung. There is also some feasibilitydata to support this approach in humans. Wholegrain FS (40 g daily) wasgiven to 2 normal volunteers for a week. Buccal swabs were taken, RNAsuccessfully extracted, and cDNA was made. The relative expressionlevels of the Nrf2-dependent gene HO-1 was measured. As shown in FIG.45, HO-1 mRNA expression was increased by 34% in one subject and 63% inthe second (p≦0.05). These data show this approach is feasible and thatFS and SDG boost Nrf2-dependent genes in normal healthy volunteers.

In the trial, buccal swabs are taken at baseline and after 2 & 4 weeksof the fiber control and FS diet, in both the control and smokingsubjects. mRNA and cDNA are generated as above and subjected to RT-PCRto evaluate the relative expression levels of representative antioxidantand Phase II drug metabolizing enzymes, including NQO-1, HO-1, andglutathione-S-transferases (GST). In addition to being “classic”representatives of Nrf2-inducible enzymes, the selected enzymes may alsoplay important mechanistic roles. NQO1 plays a dual role in thedetoxification and activation of pro-carcinogens that are present intobacco smoke. Variant genotypes of NQO1 were significantly associatedwith decreased risk of lung cancer in Japanese subjects. HO-1 has alsobeen implicated as a cytoprotective agent against oxidants and aromatichydrocarbons in cigarette smoke in genetic studies. Therefore, HO-1 alsorepresents a significant biomarker for monitoring the effects offlaxseed. Numerous studies have shown relationships between GSTpolymorphisms, smoking, and lung cancer. Importantly,benzo[a]pyrene-derived DNA-adducts in lung cells are regulated bydetoxification of the reactive intermediate resulting from bothcytochrome P-450- and aldo-keto-reductase-mediated metabolism by alung-specific GST. As benzo[a]pyrene is a significant lung carcinogenand it is present in tobacco smoke, GSTs are monitored as biomarkers ofthe effects of flaxseed in smokers.

Data Analysis:

A large pool of cDNA derived from buccal swabs of 5 normal, non-smokingvolunteers is made to be used as the “baseline” comparator for allanalyses. The cDNA from all trial samples is normalized to this poolusing β-actin and GAPDH. Once normalized, each study sample is comparedto the baseline comparator and the percent or fold-change is calculated.The primary analysis is to compare the change in gene expression beforeand after 3 weeks of SDG ingestion in the never smokers and currentsmokers (using the paired t-test). The values are also compared to thevalues in their placebo control samples. Additional analyses comparechanges in expression with SDG across all time points, including thewashout values (repeated measures ANOVA), and comparisons betweennon-smokers and smokers at baseline and after SDG and placebo. The 2 vs3 week data and the 3 week vs the washout data is compared to establishthe kinetics of response.

Determination of Decreases in Oxidative Stress by SDG in the SamePatient Population

Although it is difficult to measure oxidant stress in human subjects,one of the most established approaches is to measure urinary levels ofIsoPs. Reliable and reproducible assays for this marker have beendeveloped. Data was obtained from patients awaiting lung transplantationwho ingested 40 g FS for up to 13 weeks. Urine IsoPs were found tosteadily decline while on the FS, an indication of systemic lowering ofoxidative stress (some IsoP subcategories ↓ down to 26% of the pre-FSlevel). Data for a representative patient are shown in FIG. 46. BaP, acarcinogen in cigarette smoke, causes reactive oxygen species-mediatedDNA strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo)formation. 8-oxo-dGuo can be detected in biological specimens (urine,liver, lung) using LC-MRM/MS. Data from 2 human volunteers indicatedthat daily consumption of 40 g FS for 3 weeks induced a trend indecrease in 8-oxo-dGuo, a trend also seen in lung transplant patients(n=5) fed the same diet (FIG. 47).

Trial:

In the trial, urine samples are taken at multiple time points, in boththe control and smoking subjects. Levels of urinary IsoPs and 8-oxo-dGuoare measured in a blinded fashion.

Exhaled Breath Condensate for Evaluating Lung Oxidative Stress:

Given that it is not feasible to perform invasive tests likebronchoscopy in these subjects, a non-invasive sample collectiontechnique, exhaled breath condensate (EBC) is used for investigatinglung oxidant stress. Many substances are found in expired breath thatare detectable in the liquid that can be obtained by cooling (i.e.,condensing) it. The advantages of this method are that it isnon-invasive and convenient, as a non-invasive sampling method for thereal-time analysis and evaluation of oxidative stress biomarkers inlung. Biomarkers of oxidative stress include: H₂O₂, isoprostanes(IsoPs), malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathioneand nitrosative stress markers. Isoprostane levels in EBC are evaluatedas a useful noninvasive approach to study the effect of FS on lungoxidative stress. 8-isoPs was measure from EBCs collected from controlsubjects and smokers. The levels in non-smokers were uniformly low-nearthe sensitivity of the test (1.2 pg/ml+/−0.6, n=8), suggesting that EBCis not useful in non-smokers. Although levels were only measured in foursmokers, interestingly, two of these subjects had much higher levels: 16pg/ml and 6.5 pg/ml. Thus, in those smokers who have high baselinelevels, EBC is used to follow lung specific oxidative stress.

Data Analysis.

The baseline value for each subject is determined and the change seen atend of treatment (3 weeks) is evaluated using a paired t-test.Additional analyses compare changes in expression with SDG across antime points, including the washout values (repeated measures ANOVA), andcomparisons between non-smokers and smokers at baseline and after SDG.We can compare the 2 vs 3 week data and the 3 week vs the washout datato establish the kinetics of response.

The Chemopreventive Properties of the Flaxseed Lignan, SDG

In these experiments, the hypothesis that SDG can reduce the toxicitiesfrom xenobiotics and detoxify of carcinogens, such as BaP, similarly toSulforaphane is tested. First, the in vitro effects of SDG on bronchialepithelial cells are evaluated. Cytotoxicity tests, such as MTT assay,are conducted for an SDG doses described below. Second, SDG given orallyin the BaP mouse model is evaluated.

Evaluation of SDG-Mediated Detoxification of BaP in Normal Mouse andHuman Primary Lung Epithelial Cells and Induction of Nrf2-Mediated,Phase II Enzyme Expression.

BaP (10 or 20 μM) induces ROS in a dose- and time-dependent manner whenmetabolically activated by epithelial cells (FIG. 23). For mousestudies, the immortalized C10 mouse bronchial cell line (derived fromBalb/C mice) are used given that these cells are non-tumorigenic,contact-inhibited, and contain an the Nrf2-regulating machinery underinvestigation. Similar studies are performed using A549 cells (atransformed, but highly differentiated lung cancer cell line used tomodel human type 1 alveolar epithelial cells—see data in FIGS. 22-23,and importantly, primary human bronchial epithelial cells.

To evaluate direct ROS interception, a range of clinically-relevant, SDGconcentrations (0.1-10 μM) is added at the same time as 10 μM B[a]P andis measured for decreases in H₂DCF fluoresence, signifying directanti-oxidant activity. Liquid chromatography/multiple reactionmonitoring mass spectrometry (LC/MRM-MS) is also to determine theGSSG:GSH ratio. BaP can also quickly induce DNA adduct formation thatcan be measured using LC-MRM/MS (FIG. 22). BaP-induced oxidative DNAdamage caused by BaP can be measured by 8-oxo-dGuo formation, tailmoment of individual cells using standard COMET image analysis (data notshown), or densitometric analysis of immunoblots for the detection ofγH2AX (data not shown). To evaluate DNA adduct and oxidative changes, arange of SDG concentrations is added at the same time as 10 μM B[a]P andthese parameters are measured. Additionally, MTT cytotoxicity assays areperformed to evaluate direct SDG effects on these cells in the absenceand presence of the carcinogen (FS and FLC can only be tested in vivo).Next, the ability of the purified SDG to upregulate Phase II enzymes inbronchial epithelial cells is tested. Specifically, the induction ofGST-Ya, and NQO-1 message and protein after 0, 1, 2, 4, 24, 48, and 72hours of incubation with the lignan SDG (0.1-10 μM) treatment ismeasured. These enzymes are selected as characteristic representativeARE-regulated enzymes of the Phase II system. RT-PCR and immunoblotanalyses are carried out. After identifying the time course of Phase IIenzyme upregulation, the epithelial cells are pretreated with SDG, andat the time point of maximal Phase II induction BaP is added. Oxidantstress, DNA damage, and cell death is assessed using the assaysdescribed above.

Data analysis, Expected Results and additional studies: The first set ofstudies define the direct antioxidant effects of SDG. The second set ofstudies, performed at the later time points when the SDG itself is gone(thus no direct antioxidant effects present), define the effects of SDGdue to its Phase II enzyme-inducing effects. Additional measurements ofoxidative stress and inflammation are performed to determine thedecrease by SDG of plasma oxidative stress measurements (plasmamalondialdehyde) and pro-inflammatory stress markers such as (IL-6,IL-la, IL1β, TNF-α, C-reactive protein). The chemopreventive propertiesof SDG, in the murine model of Ad mice-chemical carcinogen induced lungtumorigenesis.

The effects of orally administered SDG on progression of BaP-inducedcarcinogenesis are studied in A/J mouse lung following the same studydesign described above (FIG. 42A, Plan A). The SDG supplemented mice aredirectly compared to control. Follow-up studies are performed including:studies using SDG during only the initiation phase (FIG. 42A, Plan B),and studies using SDG during only the promotion phase (FIG. 22A, PlanC).

statistics: the animal studies generally contain more than two groups(e.g. control vs. 50 mg/Kg SDG) of 20 mice and are analyzed using ANOVAor other suitable linear models. All calculations assume a two-sidedtest, an alpha level of 0.05, and at least 80% power.

TABLE 1 Mice on 3 test groups placed on 3 experimental plans (See FIG.42A: Plans A, B, C) are sacrificed at 4, 6 and 9 months (3 time-points)i.e., 3 × 3 × 3 × 20 mice = 540 mice. Diet Groups vehicle BaP TotalsControl (0) 20 20 40  50 mg/Kg SDG 20 20 40 100 mg/Kg SDG 20 20 40TOTALS 60 60 120

Example 8 Effects of SDG on Asbestos-Induced Carcinogenesis

SDG or flaxseed diets are hypothesized to decrease asbestos inducedROS/inflammation leading to: 1) ROS, 2) decreased cytokines; 3)decreased HMGB1; 4) less tumorigenic foci; and 5) less tumors. Theunderlining hypothesis is that decreased inflammation and oxidativestress will lead to decreased malignant transformation of cells and lesstumor burden (see FIG. 48).

To test the hypothesis that the flaxseed lignan SDG will decreaseasbestos-induced inflammation, and oxidative and nitrosative stress,mouse peritoneal macrophages were exposed to varying concentrations ofcrocidolite asbestos fibers (10, 20, 30, and 40 ng/cm²). At variabletimes post asbestos exposure (3, 4, 6, and 8 hours), cells wereincubated with SDG (5004) and supernatants collected 24 hours later todetect inflammatory cytokine secretion and nitrosative/oxidative stress(FIG. 49).

SDG blunts asbestos-induced ROS secretion by human mesothelial cells invitro (FIG. 50). Experimental plan for detecting asbestos-induced ROS incells: (human mesothelial) HM cells or mouse macrophages plated in 20%DMEM for 24 hours; Added 20 μM DCF in HBSS for 1 hour; Supernatantreplaced with 1% DMEM containing H₂O₂ (100 μM), asbestos (10 μgcrocidolite/cm²) and/or SDG (50 μM); and Fluorescence level monitored upto 36 hours post-initiation of asbestos exposure. Results show thatasbestos and H₂O₂ induce high levels of ROS, however addition of SDG inthe medium significantly lowers ROS levels to baseline levels (FIG. 50).

Asbestos-Induced Oxidative Stress (ROS release) in Culture RAWMacrophages was evaluated. Cells were treated with the ROS-sensitive dyeH2DCFDA for 30 min and then they were exposed to vehicle to 40 μg/cm2asbestos fibers, or 4 uM hypochlorite solution and fluorescenceintensity was measured spectrophotometrically for 90, 150 minutes and 27hours. Asbestos-induced ROS was generated shortly post asbestos exposureand continued for the duration of the observation period (FIG. 51).

SDG given to macrophages several hours post exposure to asbestosdecreases oxidative stress (FIG. 52). Female C57/B16 mice were injectedwith 2 mL of thioglycollate and peritoneal macrophages harvested after 3days. 2 million cells per well were plated in a 6 well plate and exposedto 20 μg/cm² asbestos. Cells were treated with 25 or 50 μM SDG(synthetic SDG) 3, 4, 6, or 8 hours post-asbestos exposure. Cells wereharvested 24 hours post-asbestos exposure. Analysis performed on frozensupernatant samples. Results show that malondialdehyde, a marker forlipid peroxidation, increased over time with asbestos exposure (FIG.52A). With 25 and 50 μM SDG added to the cells, the levels of MDAsignificantly decreased (p<0.05) (FIG. 52B).

SDG Given to Macrophages Several Hours Post Exposure to AsbestosDecreases Nitrosative Stress (FIG. 53). To evaluate of SDG in bluntingnitrosative stress in cells post asbestos exposure, female C57/B16 micewere injected with 2 mL of thioglycollate and peritoneal macrophagesharvested after 3 days. 2 million cells per well were plated in a 6 wellplate and exposed to 20 μg/cm² asbestos. Cells were treated with 25 or50 μM SDG (synthetic SDG) 3, 4, 6, or 8 hours post-asbestos exposure.Cells were harvested 24 hours post-asbestos exposure. Analysis performedon frozen supernatant samples. Results show that nitrite, a marker fornitrosative stress, increased over time with asbestos exposure (FIG.53A). With 25 and 50 μM SDG added to the cells, the levels of MDA weresignificantly decreased (p≦0.05) (FIG. 53B).

SDG Given to Macrophages Several Hours Post Exposure to AsbestosDecreases inflammatory cytokine secretion (IL-1β) (FIG. 54). To evaluateSDG in blunting inflammatory cytokine secretion (IL-1β) in cells postasbestos exposure, female C57/B16 mice were injected with 2 mL ofthioglycollate and peritoneal macrophages harvested after 3 days. 2million cells per well were plated in a 6 well plate and exposed to 20μg/cm² asbestos. Cells were treated with 25 or 50 μM SDG (synthetic SDG)3, 4, 6, or 8 hours post-asbestos exposure. Cells were harvested 24hours post-asbestos exposure. Analysis performed on fresh supernatantsamples. Results show that IL-1β, an pro-inflammatory cytokine,increased over time with asbestos exposure (FIG. 54A). With 25 and 50 μMSDG added to the cells, the levels of IL1β were significantly decreased(p≦0.05) (FIG. 54B).

SDG Given to Macrophages Several Hours Post Exposure to AsbestosDecreases inflammatory cytokine secretion (TNF-α) (FIG. 55). To evaluateSDG in blunting inflammatory cytokine secretion (TNF-α) in cells postasbestos exposure, female C57/B16 mice were injected with 2 mL ofthioglycollate and peritoneal macrophages harvested after 3 days. 2million cells per well were plated in a 6 well plate and exposed to 20μg/cm² asbestos. Cells were treated with 25 or 50 μM SDG (synthetic SDG)3, 4, 6, or 8 hours post-asbestos exposure. Cells were harvested 24hours post-asbestos exposure. Analysis performed on fresh supernatantsamples. Results show that TNF-α, an pro-inflammatory cytokine,increased over time with asbestos exposure (FIG. 55A). With 25 and 50 μMSDG added to the cells, the levels of TNF-α were significantly decreased(p<0.05) (FIG. 55B).

Briefly, the in vitro experiments in this Example demonstrate that (1)SDG blocks asbestos-induced ROS in human mesothelial cells and mouse RAWmacrophages; (2) SDG blocks inflammatory cytokine secretion by mouseperitoneal macrophages exposed to asbestos; and (3) SDG blocks oxidative(lipid peroxidation) and nitrosative stress (nitrite levels) in mouseperitoneal macrophages exposed to asbestos. These in vitro experimentssupport in vivo experimentation to determine the usefulness of SDG inblunting chronic inflammation and ultimately malignancy due to asbestosexposure.

Accordingly, SDG is tested in Asbestos-Induced Mesothelioma using twomouse models, where the mice are genetically predisposed to developmesothelioma after asbestos exposure. Using these models, the acuteeffects of Flaxseed and SDG on a single dose of asbestos in the mice areevaluated, as well as testing whether Flaxseed and SDG inhibits thedevelopment of tumors in these models of accelerated, asbestos inducedMM (FIG. 56).

SDG-enriched flaxseed lignan diet (FLC) blunts asbestos-inducedabdominal inflammation in MEXTAG mice (FIG. 57). Briefly, 6 male MEXTAGmice (10-12 weeks old) were injected ip with 400 μg of crocidoliteasbestos in a volume of 0.5 mL. Half the mice were placed on a FLC dietenriched in SDG (35% SDG) for two weeks prior to asbestos injection—theothers were on standard diet. After 3 days, mice were lavaged with 5 mLof PBS and lavage taken for sups and cell counts. Cytospins were doneand 3-5 separate fields were counted for differential-% of total cellswere calculated. Compared to 0% FS fed mice, mice fed 10% FLC had a 26%reduction in abdominal lavage fluid WBCs (P=0.014) (FIG. 57).

Acute phase studies in 7 week old, male NF2 (129sv) (+/−) mice exposedto asbestos were performed to evaluate the effects of flaxseed andlignan SDG formulations administered in diets. The experimental plan ofasbestos exposure of NF2 mice and flaxseed/SDG lignan formulationevaluation is shown in FIG. 58. Briefly, Male NF2 (129SV) (+/−) wereexposed to 400 μg asbestos via intraperitoneal injection on Day 0. Micewere initiated on the test diets (0% FS, 10% FS, 10% FLC; n=2 mice pergroup) 24 hours prior to asbestos exposure (Day −1) and sacrificed onDay 3 post-asbestos. Abdominal lavage (AL) was performed using 5 mL of1×PBS. Inflammatory cell influx peaked by 3 days and tapered off by 9days post asbestos exposure. Therefore, 3 days was selected as the timepoint to evaluate inflammation in all subsequent experiments (FIG. 59).

Flaxseed and its SDG-rich lignan component blunted asbestos-inducedinflammation (younger mice) (FIG. 60). Total white blood cells (FIG.60A) decreased with FS or FLC addition in the diet, albeit notsignificantly. However, when looking at cell differentials, andmacrophage levels in particular, levels were significantly blunted byboth flaxseed and the SDG-lignan diet (FIG. 60B).

Flaxseed and its SDG-rich lignan component blunted asbestos-inducedinflammation (older mice) (FIG. 61). Older mice exposed to abdominalasbestos (FIG. 61A) are more sensitive to asbestos by presenting withapprox. 3,000,000 WBC/mL of abdominal lavage fluid as compared to just300,000 cells/mL (10-fold higher). Results indicated that theinflammatory cells Neutrophils (FIG. 61B) and Macrophages (FIG. 61C)were both significantly higher in older than in younger mice.

FIG. 62: Flaxseed lignan extract enriched in SDG (given in dietformulation) blunts asbestos inflammation in older mice (FIG. 62). MaleNF2 (129SV) (+/−) mice were injected (intraperitoneal) with 400 μg ofasbestos on Day 0. Mice were initiated on the test diets (0% FS or 10%FLC) the week prior to asbestos exposure (Day-7) and sacrificed on Day 3post-asbestos exposure. Abdominal lavage (AL) was performed with 5 mL1×PBS (1 ml of belly lavage fluid was centrifuged and the supernatantwas frozen). Plasma was collected at frozen at −80°. Cells wereevaluated in lavage fluid and showed that total WBC and neutrophils,macrophages and eosinophils were all significantly decreased by theSDG-rich diet.

Flaxseed lignan extract enriched in SDG (given in diet formulation)blunts asbestos inflammatory cytokine secretion and nitrosative stressin older mice (FIG. 63). Male NF2 (1295V)(+/−) mice were injected(intraperitoneal) with 400 μg of asbestos on Day 0. Mice were initiatedon test diets (0% FS or 10% FLC) the week prior to asbestos exposure(Day 7) and sacrificed on Day 3 post-asbestos exposure. Abdominal lavage(AL) was performed with 5 mL 1×PBS (1 mL of belly lavage fluid wascentrifuged and the supernatant frozen). Plasma was collected at frozenat −80°. Levels of cytokines IL1β and TNFα as well as nitrites weresignificantly blunted by the SDG-rich diet.

Briefly, these in vivo experiments with NF2 mice demonstrate that (1)Mice fed SDG-enriched diet had significantly reduced abdominalinflammation, as determined by abdominal lavage fluid WBCs; (2)SDG-enriched diet reduced the number of neutrophils in abdominal lavagefluid; (3) Levels of pro-inflammatory cytokines, IL-1β and TNFα, werereduced in mice fed SDG-enriched; and (4) SDG-enriched diet-fed mice hadlower levels of abdominal lavage fluid nitrite, indicative of reducednitrosative stress induced by exposure to asbestos fibers.

These studies in these Examples demonstrate SDG's chemopreventiveactivity. Larger scale biomarker studies are conducted to evaluate theinduction of Phase II enzymes in buccal epithelium and oxidative stressreduction by SDG. Additional measurements are performed measuringoxidative stress and inflammation after oral SDG daily administration incarcinogen exposed subjects, e.g. former or current smokers, and includeplasma oxidative stress measurements (plasma malondialdehyde) andpro-inflammatory stress markers such as (IL-6, IL-1α, IL1β, TNF-α,C-reactive protein, F2-isoprostanes).

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A method for protecting a biomolecule, a cell, or a tissue fromradiation damage in a subject in need thereof, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol digluco side (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.
 2. (canceled)
 3. (canceled)
 4. Themethod of claim 1, wherein the subject has been or will be exposed toradiation as part of a therapeutic procedure or as part of a diagnosticprocedure.
 5. The method of claim 4, wherein the subject is a cancerpatient who has or will receive radiotherapy.
 6. (canceled) 7.(canceled)
 8. The method of claim 4, wherein the diagnostic procedure isa dental or bone X-ray or a PET or CT scan.
 9. (canceled)
 10. The methodof claim 1, wherein the subject has been accidentally exposed toradiation.
 11. The method of claim 1, wherein the subject has been orwill be exposed to radiation as part of their occupation.
 12. (canceled)13. The method of claim 1, wherein the subject has been exposed to radonor to radiation as a result of terrorism.
 14. (canceled)
 15. A methodfor protecting a biomolecule, a cell, or a tissue fromcarcinogen-induced damage, malignant transformation and cancerdevelopment in a subject in need thereof, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.
 16. (canceled)
 17. (canceled)
 18. Themethod of claim 15, wherein the subject has cancer.
 19. (canceled) 20.The method of claim 15, wherein the subject has been or will be exposedto one or more carcinogens or chemical warfare toxicants accidentally oras a result of a terrorist act.
 21. The method of claim 15, wherein thesubject has been or will be exposed to one or more carcinogens as aresult of a habit or their occupation.
 22. The method of claim 21,wherein the habit is smoking.
 23. (canceled)
 24. The method of claim 21,wherein the subject's occupation is as a laboratory technician.
 25. Amethod for protecting a biomolecule, a cell, or a tissue from damage byhypochlorite ions in a subject in need thereof, the method comprising:administering to said subject an effective amount of at least onebioactive ingredient, wherein said bioactive ingredient comprisessecoisolaricirecinol diglucoside (SDG), secoisolariciresinol (SECO),enterodiol (ED), enterolactone (EL), analogs thereof, stereoisomersthereof, or a combination thereof.
 26. The method of claim 1, whereinthe biomolecule is a nucleic acid, a protein or a lipid.
 27. (canceled)28. The method of claim 1, wherein the bioactive ingredient is(S,S)-SDG.
 29. The method of claim 1, wherein the bioactive ingredientis (R,R)-SDG.
 30. The method of claim 1, wherein the bioactiveingredient is synthetic SDG.
 31. (canceled)
 32. (canceled) 33.(canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. A method fortreating or preventing radiation damage, hypochlorite ion-induceddamage, or carcinogen-induced damage, malignant transformation or cancerdevelopment in a subject who has been or will be exposed to radiation,to hypochlorite ions, or to one or more carcinogens, the methodcomprising: administering to said subject an effective amount of atleast one bioactive ingredient, wherein said bioactive ingredientcomprises secoisolaricirecinol diglucoside (SDG), secoisolariciresinol(SECO), enterodiol (ED), enterolactone (EL), analogs thereof,stereoisomers thereof, or a combination thereof.
 38. The method of claim37, wherein the subject has been or will be exposed to radiation as partof a therapeutic procedure or as part of a diagnostic procedure.
 39. Themethod of claim 38, wherein the subject is a cancer patient who has orwill receive radiotherapy.
 40. (canceled)
 41. (canceled)
 42. The methodof claim 38, wherein the diagnostic procedure is a dental or bone X-rayor a PET or CT scan.
 43. (canceled)
 44. The method of claim 37, whereinthe subject has been accidentally exposed to radiation or to the one ormore carcinogens.
 45. The method of claim 37, wherein the subject hasbeen or will be exposed to radiation or to the one or more carcinogensas part of their occupation.
 46. The method of claim 45, wherein thesubject's occupation is as a laboratory technician.
 47. The method ofclaim 37, wherein the subject has been exposed to radon.
 48. The methodof claim 37, wherein the subject has been exposed to radiation or to theone or more carcinogens as a result of terrorism.
 49. (canceled)
 50. Themethod of claim 37, wherein the carcinogen-induced damage, malignanttransformation or cancer development is cancer.
 51. The method of claim50, wherein the cancer is lung cancer or malignant mesothelioma. 52.(canceled)
 53. (canceled)
 54. (canceled)
 55. (canceled)
 56. The methodof claim 37, wherein the subject has been or will be exposed to one ormore carcinogens as a result of a habit.
 57. The method of claim 56,wherein the habit is smoking.
 58. (canceled)
 59. (canceled)
 60. Themethod of claim 37, wherein the one or more carcinogens is asbestos ortobacco smoke.
 61. (canceled)
 62. The method of claim 60, wherein thesubject is a smoker, a former smoker, or a non-smoker exposed to secondhand smoke.
 63. (canceled)
 64. (canceled)
 65. (canceled)
 66. The methodof claim 37, wherein the bioactive ingredient is (S,S)-SDG.
 67. Themethod of claim 37, wherein the bioactive ingredient is (R,R)-SDG. 68.The method of claim 37, wherein the bioactive ingredient is syntheticSDG.
 69. (canceled)
 70. (canceled)
 71. (canceled)
 72. The method ofclaim 37, wherein the bioactive ingredient is SDG in a concentrationabout 25 μM to about 250 μM.
 73. (canceled)
 74. The method of claim 37,wherein the subject is a human subject.
 75. A method for protecting abiomolecule, a cell, or a tissue from radiation damage or fromcarcinogen-induced damage or from damage by hypochlorite ions, themethod comprising: contacting said biomolecule, cell, or tissue with aneffective amount of at least one bioactive ingredient, wherein saidbioactive ingredient comprises secoisolaricirecinol diglucoside (SDG),secoisolariciresinol (SECO), enterodiol (ED), enterolactone (EL),analogs thereof, stereoisomers thereof, or a combination thereof. 76.(canceled)
 77. (canceled)
 78. (canceled)
 79. (canceled)
 80. The methodof claim 75, wherein the bioactive ingredient is (S,S)-SDG.
 81. Themethod of claim 75, wherein the bioactive ingredient is (R,R)-SDG. 82.The method of claim 75, wherein the bioactive ingredient is syntheticSDG.
 83. (canceled)
 84. (canceled)
 85. (canceled)
 86. (canceled) 87.(canceled)
 88. (canceled)
 89. (canceled)